Human cytomegalovirus immunogenic composition

ABSTRACT

The invention relates to an immunogenic composition comprising an HCMV gB antigen, an HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen and a Th1-inducing adjuvant. If further relates to the immunogenic composition for use as an HCMV vaccine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage application under 35 U.S.C. §371 of International Application No. PCT/EP2018/074369, filed on Sep.11, 2018, which claims priority to European Patent Application No.17306179.7, filed on Sep. 13, 2017, the entire contents of each of whichare incorporated herein by reference.

The invention relates to an immunogenic composition comprising an HCMVgB antigen, an HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigenand a Th1-inducing adjuvant. It further relates to the immunogeniccomposition for use as an HCMV vaccine.

BACKGROUND OF THE INVENTION

The Human cytomegalovirus (HCMV) is a ubiquitous virus belonging to theHerpes virus family. The virus is composed of a linear double-strandeddeoxyribonucleic acid (DNA) contained in a capsid surrounded by ategument and enveloped in a lipid bilayer carrying glycoprotein spikeson its surface. Like other members of this family, HCMV possesses thecharacteristics of latency and reactivation. HCMV has the ability toinfect and be latent in many cells.

In the immunocompetent host, most HCMV infections are asymptomatic orvery mild with a few nonspecific symptoms such as fatigue, malaise,moderate fever, lymphadenopathy, hepatomegaly or a slight increase inliver enzymes. Heterophil-negative mononucleosis is however observed inapproximately 10% of previously healthy individuals.

In contrast, clinical manifestations can be very severe in newbornsinfected in utero and in adults immunosuppressed by AIDS or in thecontext of solid organ or bone marrow transplantation.

The prevalence of HCMV infection increases with age and is affected bysocioeconomic factors. Serological surveys have shown a higherprevalence in developing countries and in lower socioeconomic groups ofdeveloped countries. For women of child-bearing age, the proportion ofHCMV seropositive women ranges from approximately 50% in upper andmiddle incomes groups of developed countries to over 80% in low-incomepopulations. Surveys performed in different western European countrieswithin the two last decades on the general population includingdifferent age-classes, females and males showed globally that HCMVseroprevalence in toddlers and adolescents ranges between 40 and 50%while in older subjects (40 years and over), HCMV seroprevalence ishigher than 80%.

HCMV is shed for a prolonged period in the secretions of infectedindividuals including urine, saliva, milk, semen, genital secretions;HCMV is thus transmitted either horizontally (through intimate contactfrom child to child, from child to parents and between sex partners) orvertically from mother to fetus or infant through the placenta or atbirth through body fluids contacts and breast feeding or by exposure toblood products or transplanted organs.

HCMV is the most common cause of congenital infection in the developedworld. Congenital infection refers to infection transmitted from motherto fetus prior to birth of the newborn. Each year in the United States,an estimated 8000 infants suffer disabilities, including mentalretardation, blindness and sensorineural deafness, as a result ofcongenital HCMV infection.

Among congenitally infected newborns, 5% to 10% have majormanifestations at birth such as microcephaly, chorioretinitis,intracranial calcifications, hepatosplenomegaly, hepatitis, jaundice,direct hyperbilirubinemia, thrombocytopenia, petechiae, and anemia.Among these newborns with symptomatic congenital HCMV disease, themortality rate is approximately 10% in early infancy and amongsurvivors, 50-90% will have sequelae such as mental retardation,cerebral palsy, sensorineural hearing loss or visual impairment.

Many infants with congenital HCMV infection are asymptomatic at birth.Follow-up studies have shown that approximately 15% of infants who areasymptomatic at birth and identified as HCMV seropositive in the newbornperiod by virological screening will have sequelae such as hearing lossor central nervous system abnormalities.

As a whole, approximately 17,000 infants born each year in Europe and inthe USA will have permanent sequelae.

Congenital HCMV infections are more frequent and more severe when theprimary infection occurs in the first trimester of pregnancy than whenprimary infection occurs later in pregnancy. Overall, a primary HCMVinfection during pregnancy is associated with a 40% risk of transmissionto the fetus.

Effective means of preventing or treating maternal HCMV infection duringpregnancy or congenital HCMV infection are currently not available.

HCMV is also an important viral pathogen in organ and bone marrowtransplant recipients and in AIDS patients. The rate of HCMV-associatedmorbidity in HCMV seronegative solid organ transplant recipientsapproaches 60%. In solid organ transplant the disease is the most severewhen seronegative patients receive a graft from a HCMV positive donor.In contrast, in bone marrow or stem cell transplantation the disease ismost severe in HCMV seropositive subjects receiving cells from aseronegative donor showing that the origin of HCMV infection isreactivation of endogenous infection.

HCMV causes pneumonitis, hepatitis, gastrointestinal disease, bonemarrow suppression, and retinitis in approximately 15% of allograftrecipients. In addition to these direct end-organ diseases, HCMV hasbeen associated with indirect effects such as graft rejection,accelerated atherosclerosis and immunosuppression that can lead tobacterial or fungal infection.

Development of an HCMV vaccine is therefore considered a major publichealth objective in Institute of Medicine vaccine prioritization reports(Kathleen R, Stratton, Jane S, Durch, Lawrence R S. Editors committee tostudy priorities for Vaccine Development Division of Health Promotionand Disease Prevention Institute of Medicine. In: Vaccines for the 21stcentury: A tool for decision making. Washington D.C.: National AcademyPress; 2000). Many candidate vaccines have been described, but, so far,none has been licensed (Plotkin et al., Vaccines, 6^(th) edition, Ed.Elsevier, 2013, Schleiss et al., Cytomegalovirus vaccines, pages1032-1041).

A cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant showedpromising results in a phase 2 randomised placebo-controlled trial intransplant recipients (Griffiths et al., Lancet, 2011,377(9773):1256-63). A phase 2, placebo-controlled, randomized,double-blind trial in women of child-bearing age, evaluated the samevaccine consisting of recombinant HCMV envelope glycoprotein B with MF59adjuvant, as compared with placebo. The results showed 50% efficacy inpreventing HCMV acquisition of primary HCMV. However the immunogenicityresults showed that the level of neutralizing antibodies (Ab) induced bythe gB/MF59 formulation are at the peak level one month after theadministration of the 3rd dose, and then rapidly decline (Pass et al.,The New England Journal of Medecine, 2009, 360:1191-9).

As a consequence, there is a need to improve the HCMV vaccine efficacy,in particular to find a vaccine that increases neutralizing antibodylevels and induces long-lasting protection by inducing persistent immuneresponse. There is also a need to find a HCMV vaccine that moreparticularly induces a broader immune response.

DESCRIPTION OF THE INVENTION

Unexpectedly, the inventors of the present invention have now found anew immunogenic composition that complies with these requirements.

The present invention thus relates to an immunogenic compositioncomprising an HCMV gB antigen, an HCMV gH/gL/UL128/UL130/UL131pentameric complex antigen and a Th1-inducing adjuvant.

In particular, said Th1-inducing adjuvant comprises:

-   -   a TLR-4 agonist; or    -   a polyacrylic acid polymer salt with a weight average molecular        weight Mw in the range of 350 to 650 kDa.

In one embodiment, said Th1-inducing adjuvant comprises a TLR-4 agonist.

In particular, said TLR4 agonist is in combination with a deliverysystem such as aqueous nanosuspension, calcium phosphate, liposomes,virosomes, ISCOMs, micro- and nanoparticles, or emulsions.

More particularly, said delivery system is an oil-in-water emulsion.

In particular, said TLR-4 agonist is chosen from E6020 (CAS number:287180-63-6) and a GLA (CAS Number 1246298-63-4) TLR-4 agonist.

In one embodiment, said Th1-inducing adjuvant comprises a linear orbranched polyacrylic acid polymer salt with a weight average molecularweight Mw in the range of 350 to 650 kDa, in particular PAA225000.

In particular, said HCMV gB antigen comprises one or several mutationsat the endoproteolytic cleavage site.

Still particularly, said HCMV gB antigen is a full length gBpolypeptide, a full length gB polypeptide lacking at least a portion ofthe transmembrane domain, a full length gB polypeptide lackingsubstantially all the transmembrane domain, a full length gB polypeptidelacking at least a portion of the intracellular domain, a full length gBpolypeptide lacking substantially all the intracellular domain, or afull length gB polypeptide lacking substantially both the transmembranedomain and the intracellular domain.

More particularly, said HCMV gB antigen is gBdTm.

In particular, in the said HCMV gH/gL/UL128/UL130/UL131 pentamericcomplex antigen, the gH antigen lacks at least a portion of thetransmembrane domain, preferably the gH antigen lacks substantially allthe transmembrane domain.

More particularly, said gH comprises the ectodomain of the full lengthgH encoded by UL75 gene.

Still particularly, in the immunogenic composition according to theinvention, the HCMV gB and the HCMV gH/gL/UL128/UL130/UL131 pentamericcomplex are the sole HCMV antigens.

The present invention further relates to the immunogenic compositionaccording to the invention for use as an HCMV vaccine.

In particular, said vaccine increases neutralizing antibody levelsand/or persistence.

Immunogenic Composition

As previously mentioned, the immunogenic composition according to theinvention comprises:

-   -   an HCMV gB antigen;    -   an HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen; and    -   a Th1-inducing adjuvant.

“HCMV” is used for Human cytomegalovirus and is any strain of Humancytomegalovirus.

The terms “comprising”/“comprises”/“comprise”/“comprised” encompass“including”/“includes”/“include”/“included” respectively as well as“consisting”/“consists”/“consist”/“consisted” respectively, e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional, e.g. X+Y.

“Antigen” as used herein, has the common meaning known by a man skilledin the art. In particular, it refers to any molecule containing one ormore epitopes (either linear, conformational or both), that elicits animmunological response.

In the context of the present invention, an antigen further includes aprotein having modifications, such as deletions, additions andsubstitutions to the native sequence, as long as the protein maintainssufficient immunogenicity. These modifications may be deliberate, forexample through site-directed mutagenesis, or may be accidental, such asmutations which occur during expression of the antigens in a host cell.The antigen may also be a protein or a fragment thereof encoded by aconsensus sequence.

The antigen(s) which can be used in an immunogenic composition accordingto the invention are in particular an HCMV gB antigen and an HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen.

HCMV gB Antigen

The HCMV gB antigen according to the present invention is a full lengthgB polypeptide or a gB-derived polypeptide that induces neutralizingantibodies.

gB is encoded by the UL55 gene of HCMV genome. The size of the nativeform of gB (or gp130) depends on the size of the open reading frame(ORF), which may vary a little according to the strain. For example, theORF of AD169 strain, which is 2717 bp long, encodes a full length gB of906 amino acids whereas the ORF of Towne strain encodes a full length gBof 907 amino acids. The protein sequences of these two strains aredescribed in US 2002/0102562 (FIG. 2), incorporated by reference in itsentirety. The native form of gB contains an amino acid signal sequencethat is normally 23 to 25 amino acid long, followed by an extracellulardomain containing an endoproteolytic cleavage site between residuesarginine 460 and serine 461, by a transmembrane domain and by anintracellular domain. Usually, the full length gB is depleted of theamino acid signal sequence as a consequence of posttranslationalmechanisms that occur in cells. It will be well understood that suitablefull length gB for the purpose of the invention encompasses both thefull length gB of HCMV strains Towne and AD169, as well as otherequivalent strains. Several antigenic domains inducing neutralizingantibodies have been described. Notably, it includes the domain that islocated between amino acid residues 461 and 680 of gp 130, this domainbeing subdivided into two discontinuous domains, the domain betweenresidues 461 and 619 and the domain between residues 620 and 680 (U.S.Pat. No. 5,547,834). It also includes the antigenic domain 1 (AD-1)located between amino acid residues 560 and 640 (Schoppel K. et al.,Virology, 1996, 216:133-45) or the antigenic domain 2 (AD-2) locatedbetween amino acid residues 65 and 84 (Axelsson F et al., Vaccine, 2007,26:41-6) or between amino acid residues 27 and 84 (Burke H G et al.,PLoS pathogens, 2015, 11:e1005227). Consequently, a polypeptide thatcomprises in its amino acid sequence a sequence homologous to one orseveral of the above cited antigenic domains is also suitable for thepurpose of the invention. The term “a sequence homologous to” isintended to mean an amino acid sequence in which there is at least 80%identity with the amino acid sequence of the antigenic domain beingconsidered of the native gB originating from the Towne or AD169 strain(which are described in US 2002/0102562). Typically, the sequencehomology is based on a sequence identity of at least 90% and, even morespecifically, the sequence homology is complete (sequence identity of100%).

As used herein, a first sequence having at least x % identity with asecond sequence means that x % represents the number of amino acids inthe first sequence which are identical to their matched amino acids ofthe second sequence when both sequences are optimally aligned via aglobal alignment, relative to the total length of the second amino acidsequence. Both sequences are optimally aligned when x is maximum. Thealignment and the determination of the percentage of identity may becarried out manually or automatically using a global alignmentalgorithm, for instance the Needleman and Wunsch algorithm, described inNeedleman and Wunsch, J. Mol Biol., 48, 443-453 (1970), with for examplethe following parameters for polypeptide sequence comparison: comparisonmatrix: BLOSUM62 from Henikoff and Henikoff, Proc. Natl. Acad. Sci.USA., 89, 10915-10919 (1992), gap penalty: 8 and gap length penalty: 2;and the following parameters for polynucleotide sequence comparison:comparison matrix: matches=+10, mismatch=0; gap penalty: 50 and gaplength penalty: 3.

A program which may be used with the above parameters is publiclyavailable as the “gap” program from Genetics Computer Group, MadisonWis. The aforementioned parameters are the default parametersrespectively for peptide comparisons (along with no penalty for endgaps) and for nucleic acid comparisons.

Among the gB-derived peptides or polypeptides that are suitable for theobject of the invention is gp 55 as described in U.S. Pat. No.5,547,834. It is derived from the cleavage of gB at the endoproteolyticcleavage site; its amino acid sequence corresponds to that which isbetween serine residue 461 and the C-terminal end. Truncated forms of gp55 can also be used, such as a gp 55 depleted of all or part of thetransmembrane sequence and of all or part of the intracellularC-terminal domain (for example, a peptide having a sequence homologousto the amino acid sequence of the native gB between residues 461 and646) or a gp 55 depleted of all or part of the intracellular C-terminaldomain (for example, a peptide having a sequence homologous to the aminoacid sequence of the native gB between residues 461 and 680). Suchtruncated forms of gp 55 are also described in U.S. Pat. No. 5,547,834,incorporated by reference in its entirety.

It is also possible to use a mutated form of the full length gB thatcarries one or several mutations at the endoproteolytic cleavage sitesuch that the latter is made ineffectual. In particular, the mutation(s)is (are) located between residues 457 and 460 of the sequence of gp130and, more particularly, are located at arginine 460 and/or lysine 459and/or arginine 457. In this aspect, the mutated form of the full lengthgB carries the entire extracellular domain with all the domains that aretargets for neutralizing antibodies. Such mutated forms can besecondarily depleted of all or part of the transmembrane sequence and/orof all or part of the intracellular C-terminal domain in order to allowtheir secretion in the host when produced as recombinant proteins andtheir easy downstream purification. Such gB-derivatives are preferred inso far as substantially all the domains that are targets forneutralizing antibodies are conserved.

Therefore, in one aspect of the invention the HCMV gB comprises one orseveral mutations on the endoproteolytic cleavage site, and inparticular the HCMV gB is in addition selected from among the group of afull length HCMV gB, a full length HCMV gB lacking at least a portion ofthe transmembrane domain, a full length gB polypeptide lackingsubstantially all the transmembrane domain, a full length gB polypeptidelacking at least a portion of the intracellular domain, a full lengthHCMV gB lacking substantially all the intracellular domain, and a fulllength HCMV gB polypeptide lacking substantially both the transmembranedomain and the intracellular domain.

The expression “lacking substantially all the intracellular domain” or“lacking substantially all the transmembrane domain” means that at least80% of the amino acid sequence corresponding to the said domain isdeleted.

In the context of the present invention, by “lacking at least a portionof a domain”, is meant lacking at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60% or at least 70% but lackingless than 80% of the domain.

In one embodiment, the HCMV gB antigen is the ectodomain of gB, i.e. afull length gB depleted of all the transmembrane sequence and of all theintracellular C-terminal domain. The “ectodomain” is the portion of atransmembrane anchored protein that extends beyond the membrane into theextracellular space.

The HCMV gB antigen according to the present invention may also containother mutations and/or deletions and/or additions. For instance, theHCMV gB antigen may contain at least one amino acid deletion orsubstitution in at least one of the fusion loop 1 (FL1) domain andfusion loop 2 (FL2) domain located in the extracellular domain asdescribed in EP2627352. Alternatively or in addition, it may contain adeletion of at least a portion of the leader sequence as described inEP2627352. The HCMV gB antigen according to the present invention mayalso comprise a mutation that results in a glycosylation site withinhydrophobic surface 1 (amino acid residues 154-160 and 236-243) asdescribed in WO2016092460. In particular, said glycosylation site is anN-glycosylation site comprising an N—X—S/T/C motif, wherein X is anyamino acid residue (but preferably not proline). The HCMV gB antigen maycomprise a mutation that results in a glycosylation site, wherein saidglycosylation site is (1) within hydrophobic surface 2 (amino acidresidues 145-167 and 230-252); or (2) at a residue that is within 20angstroms from fusion loop 1 (FL1) (amino acid residues 155-157) and/orfusion loop 2 (FL2) (amino acid residues 240-242), as described inWO2016092460. The HCMV gB antigen may comprise a heterologous sequencethat is at least 12 residues long at the C-terminus as described inWO2016092460. In particular, the gB protein may be a fusion proteinwherein the heterologous sequence is fused at the C-terminus of theectodomain.

Native HCMV gB has been postulated to be a homotrimer based on the 3Dcrystallography structure of gB proteins in related viruses, HerpesSimplex Virus 1 (HSV-1) gB and Epstein Barr Virus (EBV) gB, which arehomotrimers (Heldwein et al., Science, 2006, 313:217-220; Backovic etal., PNAS, 2009, 106(8):2880-2885). The HCMV gB antigen according to thepresent invention may be in a trimeric (native form), and/or hexameric(dimer of the trimeric native form), and/or dodecameric (dimer ofhexamer) form. In particular, the HCMV gB antigen part of theimmunogenic composition according to the present invention issubstantially not in a monomeric form, more particularly not in amonomeric form. The expression “is substantially not in a monomericform” means that less than 20%, in particular less than 10%, inparticular less than 5%, of the HCMV gB antigen is in a monomeric form.

According to an embodiment, the gB antigen comprises an amino acidsequence which has at least 80% identity with SEQ ID NO: 1. Inparticular, said gB antigen comprises an amino acid sequence which hasat least 85% identity, at least 90% identity, at least 95% identity, atleast 97% identity, at least 98% identity, at least 99% identity or even100% identity with SEQ ID NO: 1:

SSTRGTSATHSHHSSHTTSAAHSRSGSVSQRVTSSQTVSHGVNETIYNTTLKYGDVVGVNTTKYPYRVCSMAQGTDLIRFERNIVCTSMKPINEDLDEGIMVVYKRNIVAHTFKVRVYQKVLTFRRSYAYIHTTYLLGSNTEYVAPPMWEIHHINSHSQCYSSYSRVIAGTVFVAYHRDSYENKTMQLMPDDYSNTHSTRYVTVKDQWHSRGSTWLYRETCNLNCMVTITTARSKYPYHFFATSTGDVVDISPFYNGTNRNASYFGENADKFFIFPNYTIVSDFGRPNSALETHRLVAFLERADSVISWDIQDEKNVTCQLTFWEASERTIRSEAEDSYHFSSAKMTATFLSKKQEVNMSDSALDCVRDEAINKLQQIFNTSYNQTYEKYGNVSVFETTGGLVVFWQGIKQKSLVELERLANRSSLNLTHNTTQTSTDGNNATHLSNMESVHNLVYAQLQFTYDTLRGYINRALAQIAEAWCVDQRRTLEVFKELSKINPSAILSAIYNKPIAARFMGDVLGLASCVTINQTSVKVLRDMNVKESPGRCYSRPVVIFNFANSSYVQYGQLGEDNEILLGNHRTEECQLPSLKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVLELYSQKELRSSNVFDLEEIMREFNSYKQRVKYVEDKRLCMQPLQNLFPYLVSADGTTVTSGNTKDTSLQAPPSYEESVYNSGRKGPGPPSSDASTAAPPYTNEQAYQMLLALVRLDAEQRAQQNGTDSLDGQTGTQDKGQKPNLLDRLRHRKNGYRHLKDS DEEENV.

In a preferred embodiment the gB antigen comprises an amino acidsequence which has 100% identity with SEQ ID NO: 1.

An HCMV gB antigen that is particularly suitable in the context of thepresent invention is a truncated form of the full length gB depleted ofall or part of the C-terminal domain and/or depleted of all or part ofthe transmembrane sequence and in which the cleavage site isineffectual. A truncated form of gB that is particularly preferredcorresponds to that which is described in U.S. Pat. No. 6,100,064,called gBdTM, incorporated by reference in its entirety. In U.S. Pat.No. 6,100,064 the signal sequence was hypothetized as 24 amino acidslong and the amino acid positions were indicated accordingly on FIG. 10. The inventors have discovered that this signal sequence is in fact 25amino acids long. In FIG. 10 of U.S. Pat. No. 6,100,064 all the aminoacid positions indicated C-terminal of the Ser-1 (i.e. C-terminal to thesignal sequence) have so to be decreased from 1. Accordingly gBdTMcarries three mutations at the cleavage site (Arginine 432 issubstituted by Threorine, Lysine 434 is substituted by Glutamine andArginine 435 is substituted by Threonine; taking into account therenumbered positions) and a deletion in the transmembrane region betweenamino acid residues valine 676 and arginine 751 (taking into account therenumbered positions), such that the extracellular domain is directlyconnected to the cytoplasmic domain. Such gB-derived polypeptide iseasier to purify as it is produced by recombinant cells expressing thisproduct under a secreted form. The resulting form is an 806 amino acidlong polypeptide deleted of its signal sequence and of its transmembraneregion when it is derived from the gB Towne strain.

The HCMV gB protein described herein or the peptides or polypeptidesderived therefrom may be synthesized by any method well-known to the manskilled in the art. Such methods include conventional chemicalsynthesis, in solid phase (R. B. Merrifield, J. Am. Chem. Soc., 85 (14),2149-2154 (1963)), or in liquid phase, enzymatic synthesis (K. Morihara,Trends in Biotechnology, 5(6), 164-170 (1987)) from constitutive aminoacids or derivatives thereof, cell-free protein synthesis (Katzen etal., Trends in Biotechnology, 23(3), 150-156 (2005)), as well asbiological production methods by recombinant technology.

For example, the HCMV gB antigen can be obtained using a biologicalproduction process with a recombinant host cell. In such a process, anexpression cassette, containing a nucleic acid encoding an HCMV gBantigen as described herein, is transferred into a host cell, which iscultured in conditions enabling expression of the corresponding protein.The protein thereby produced can then be recovered and purified. Methodsfor the purification of proteins are well-known to the skilled person.The obtained recombinant protein can be purified from lysates and cellextracts or from the culture medium supernatant, by methods usedindividually or in combination, such as fractionation, chromatographicmethods, immunoaffinity methods using specific mono- or polyclonalantibodies, etc. In particular, the obtained recombinant protein ispurified from the culture medium supernatant.

The HCMV gB protein or the peptides or polypeptides derived therefromare usually obtained by recombinant DNA techniques and purifiedaccording to methods well known to those skilled in the art. The methodsdescribed in U.S. Pat. No. 6,100,064 and in US 2002/0102562,incorporated by reference in their entirety, can in particular be used.

For example, the gB antigen according to the invention is a recombinantglycoprotein, which is produced in Chinese hamster ovary (CHO) cellcultures. The gB gene from the Towne strain of HCMV can be mutagenizedto remove the cleavage site and the transmembrane portion of themolecule in order to facilitate secretion in cell culture as describedin U.S. Pat. No. 6,100,064. The secreted molecule is a polypeptide of806 amino acids, retaining 19 potential N-linked glycosylation sites,and is also called gBdTm. The purification process involved affinity andion-exchange chromatography steps.

HCMV gH/gL/UL128/UL130/UL131 Pentameric Complex Antigen

Another antigen part of the immunogenic composition according to theinvention is the HCMV gH/gL/UL128/UL130/UL131 pentameric complexantigen.

Said pentameric complex is assembled through disulfide bonds andnon-covalent interactions among the five components to form a functionalcomplex able to present conformational epitopes (Ciferri et al., PNAS,2015, 112(6):1767-1772; Wen et al., Vaccine, 2014, 32(30):3796-3804).

Said complex has already been described and is known by the man skilledin the art. It is in particular described in Ryckman et al. (Journal ofVirology, January 2008, p. 60-70) and in patent applicationWO2014/005959. Said HCMV gH/gL/UL128/UL130/UL131 pentameric complex canin particular comprise a modified HCMV gH polypeptide, wherein saidpolypeptide lacks at least a portion of the transmembrane (TM) domain.In some embodiments, the gH polypeptide can retain a portion of thenatural TM domain, but not enough to let the protein stay in a lipidbilayer. In a preferred embodiment the gH polypeptide lackssubstantially all the transmembrane domain. In a more preferredembodiment the gH polypeptide lacks the full-length natural TM domain.

Thus, the gH polypeptide can contain up to 10 amino acids (e.g. 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 amino acids) of the natural gH TM domain.

In the context of the present invention, by “lacking at least a portionof a domain”, is meant lacking at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60% or at least 70% but lackingless than 80% of the domain.

The expression “lacking substantially all the intracellular domain” or“lacking substantially all the transmembrane domain” means that at least80% of the amino acid sequence corresponding to the said domain isdeleted.

Alternatively or in addition to lacking a portion or all of the TMdomain, the polypeptide may lack a portion or substantially all or allthe intracellular domain of HCMV gH.

In a preferred embodiment the gH polypeptide lacks substantially all theintracellular domain. In a more preferred embodiment the gH polypeptidelacks the full-length natural intracellular domain.

In a preferred embodiment, the gH polypeptide lacks all the TM domainand all the intracellular domain.

In one embodiment, said gH comprises the ectodomain of the full lengthgH encoded by UL75 gene.

HCMV glycoprotein H (gH), which is encoded by the UL75 gene, is a virionglycoprotein that is essential for infectivity and which is conservedamong members of the alpha-, beta- and gamma-herpes viruses. It forms astable complex with gL, and the formation of this complex facilitatesthe cell surface expression of gH. Based on the crystal structures ofHSV-2 and EBV gH/gL complexes, the gL subunit and N-terminal residues ofgH form a globular domain at one end of the structure (the ‘head’),which is implicated in interactions with gB and activation of membranefusion. The C-terminal domain of gH, proximal to the viral membrane (the‘tail’), is also implicated in membrane fusion. In one embodiment, thegH polypeptide in the pentameric complex described herein comprises anamino acid sequence which has at least 80% identity with SEQ ID NO: 2.In particular, the gH antigen comprises an amino acid sequence which hasat least 85% identity, at least 90% identity, at least 95% identity, atleast 97% identity, at least 98% identity, at least 99% identity or even100% identity with SEQ ID NO: 2:

RYGAEAVSEPLDKAFHLLLNTYGRPIRFLRENTTQCTYNNSLRNSTVVRENAISFNFFQSYNQYYVFHMPRCLFAGPLAEQFLNQVDLTETLERYQQRLNTYALVSKDLASYRSFSQQLKAQDSLGEQPTTVPPPIDLSIPHVWMPPQTTPHGWTESHTTSGLHRPHFNQTCILFDGHDLLFSTVTPCLHQGFYLIDELRYVKITLTEDFFVVTVSIDDDTPMLLIFGHLPRVLFKAPYQRDNFILRQTEKHELLVLVKKDQLNRHSYLKDPDFLDAALDFNYLDLSALLRNSFHRYAVDVLKSGRCQMLDRRTVEMAFAYALALFAAARQEEAGAQVSVPRALDRQAALLQIQEFMITCLSQTPPRTTLLLYPTAVDLAKRALWTPNQITDITSLVRLVYILSKQNQQHLIPQWALRQIADFALKLHKTHLASFLSAFARQELYLMGSLVHSMLVHTTERREIFIVETGLCSLAELSHFTQLLAHPHHEYLSDLYTPCSSSGRRDHSLERLTRLFPDATVPATVPAALSILSTMQPSTLETFPDLFCLPLGESFSALTVSEHVSYVVTNQYLIKGISYPVSTTVVGQSLIITQTDSQTKCELTRNMHTTHSITAALNISLENCAFCQSALLEYDDTQGVINIMYMHDSDDVLFALDPYNEVVVSSPRTHYLMLLKNGTVLEVTDVVVDATDSR.

In a preferred embodiment the gH polypeptide comprises an amino acidsequence which has 100% identity with SEQ ID NO: 2.

HCMV glycoprotein L (gL) is encoded by the UL115 gene. gL is thought tobe essential for viral replication and all known functional propertiesof gL are directly associated with its dimerization with gH. The gL/gHcomplex is required for the fusion of viral and plasma membranes leadingto virus entry into the host cell.

According to one embodiment, the gL polypeptide in the pentamericcomplex described herein comprises an amino acid sequence which has atleast 80% identity with SEQ ID NO: 3. In particular, the gL antigencomprises an amino acid sequence which has at least 85% identity, atleast 90% identity, at least 95% identity, at least 97% identity, atleast 98% identity, at least 99% identity or even 100% identity with SEQID NO: 3:

AAVSVAPTAAEKVPAECPELTRRCLLGEVFQGDKYESWLRPLVNVTGRDGPLSQLIRYRPVTPEAANSVLLDEAFLDTLALLYNNPDQLRALLTLLSSDTAPRWMTVMRGYSECGDGSPAVYTCVDDLCRGYDLTRLSYERSIFTEHVLGFELVPPSLFNVVVAIRNEATRTNRAVRLPVSTAAAPEGITLFYGLYNAVKEFCLRHQLDPPLLRHLDKYYAGLPPELKQTRVNLPAHSRYGPQAVDAR.

In a preferred embodiment the gL polypeptide comprises an amino acidsequence which has 100% identity with SEQ ID NO: 3.

According to an embodiment, the UL128 polypeptide in the pentamericcomplex described herein comprises an amino acid sequence which has atleast 80% identity with SEQ ID NO: 4. In particular, the UL128 antigencomprises an amino acid sequence which has at least 85% identity, atleast 90% identity, at least 95% identity, at least 97% identity, atleast 98% identity, at least 99% identity or even 100% identity with SEQID NO: 4:

EECCEFINVNHPPERCYDFKMCNRFTVALRCPDGEVCYSPEKTAEIRGIVTTMTHSLTRQVVHNKLTSCNYNPLYLEADGRIRCGKVNDKAQYLLGAAGSVPYRWINLEYDKITRIVGLDQYLESVKKHKRLDVCRAKMGYMLQ.

In a preferred embodiment the UL128 polypeptide comprises an amino acidsequence which has 100% identity with SEQ ID NO: 4.

UL130 is the central and the largest (214 codons) gene of the UL131A-128locus. Conceptual translation of the gene predicts a long (25 aminoacids) N-terminal signal sequence that precedes a hydrophilic proteincontaining two potential N-linked glycosylation sites (Asn85 and Asn118)within a putative chemokine domain (amino acids 46 to 120) and anadditional N-glycosylation site (Asn201) close to the end of a uniqueC-terminal region. UL130 is predicted to lack a TM domain.

It has been reported to be a luminal glycoprotein that is inefficientlysecreted from infected cells but is incorporated into the virionenvelope as a Golgi-matured form (Patrone, et al.: “HumanCytomegalovirus UL130 Protein Promotes Endothelial Cell Infectionthrough a Producer Cell Modification of the Virion.”, Journal ofVirology 79 (2005): 8361-8373).

According to an embodiment, the UL130 polypeptide in the pentamericcomplex described herein comprises an amino acid sequence which has atleast 80% identity with SEQ ID NO: 5. In particular, the UL130 antigencomprises an amino acid sequence which has at least 85% identity, atleast 90% identity, at least 95% identity, at least 97% identity, atleast 98% identity, at least 99% identity or even 100% identity with SEQID NO: 5:

SPWSTLTANQNPSPLWSKLTYSKPHDAATFYCPFIYPSPPRSPLQFSGFQRVLTGPECRNETLYLLYNREGQTLVERSSTWVKKVIWYLSGRNQTILQRMPRTASKPSDGNVQISVEDAKIFGAHMVPKQTKLLRFVVNDGTRYQMCVMKLESWAHVFRDYSVSFQVRLTFTEANNQTYTFCTHPNLIV.

In a preferred embodiment the UL130 polypeptide comprises an amino acidsequence which has 100% identity with SEQ ID NO: 5.

UL131, also called UL131A, function is required for HCMV replication notonly in endothelial cells but also in epithelial cells. According to anembodiment, the UL131A polypeptide in the pentameric complex describedherein comprises an amino acid sequence which has at least 80% identitywith SEQ ID NO: 6. In particular, the UL131A antigen comprises an aminoacid sequence which has at least 85% identity, at least 90% identity, atleast 95% identity, at least 97% identity, at least 98% identity, atleast 99% identity or even 100% identity with SEQ ID NO: 6:

QCQRETAEKNDYYRVPHYWDACSRALPDQTRYKYVEQLVDLTLNYHYDASHGLDNFDVLKRINVTEVSLLISDFRRQNRRGGTNKRTTFNAAGSLAPHAR SLEFSVRLFAN.

In a preferred embodiment the UL131 polypeptide comprises an amino acidsequence which has 100% identity with SEQ ID NO: 6.

SEQ ID NO: 2 to 6 are from the strain BE/28/2011 (Genbank ID KP745669,Kremkow et al., 2015).

In the pentameric complex antigen part of the immunogenic composition ofthe invention, gH, gL and UL128 can be linked through disulfide bonds,but UL130 and UL131A can be incorporated into the pentameric complex bynon-covalent interactions. For example, the UL130 protein and/or UL131Aprotein is incorporated into the pentameric complex by non-covalentinteractions. Furthermore, the UL130 protein and/or UL131A protein maybe inter-linked by non-covalent interactions.

A range of conformational epitopes for the pentameric complex are known.For example, Macagno (Macagno et al.: “Isolation of human monoclonalantibodies that potently neutralize human cytomegalovirus infection bytargeting different epitopes on the gH/gL/UL128-131A complex.”, Journalof Virology 84 (2010): 1005-13) isolated a panel of human monoclonalantibodies that neutralized HCMV infection of endothelial, epithelial,and myeloid cells. In one embodiment, the pentameric complex antigenpart of the immunogenic composition of the invention possesses one ormore of the conformational epitopes identified by Macagno (2010).

Each protein of the pentameric complex antigen may contain mutations,such as insertions, deletions and substitutions, so long as thesemutations are not detrimental to the use of the proteins as antigens. Inaddition, such mutations should not prevent the capacity of the proteinsto form a pentameric complex according to the invention. The ability toform a pentameric complex of the invention can be tested by performingprotein purification, and analyzing the proteins by non-reducing PAGE,Western blot and/or size exclusion chromatography. If the proteins formpart of a complex, they may all be present in a single band on a nativePAGE gel and/or be present in a single peak in a size exclusionchromatogram.

Expression of said pentameric complex can be realized according tomethods known by the man skilled in the art. Mention can be made forexample of the method described in Hofmann et al. (Biotechnology andBioengineering, 2015).

Suitable expression systems for use in the context of the presentinvention are well known to the man skilled in the art and many aredescribed in detail in Doyle (Doyle, ed. High Throughput ProteinExpression and Purification: Methods and Protocols (Methods in MolecularBiology). Humana Press, 2008). Generally, any system or vector that issuitable to maintain, propagate and express nucleic acid molecules toproduce a polypeptide in the required host may be used. The appropriatenucleotide sequence may be inserted into an expression system by any ofa variety of well-known and routine techniques, such as, for example,those described in Sambrook (Sambrook, J. Molecular Cloning: ALaboratory Manual. 3rd. Cold Spring Harbor Laboratory Press, 2000).Generally, the encoding gene can be placed under the control of acontrol element such as a promoter, and, optionally, an operator, sothat the DNA sequence encoding the desired peptide is transcribed intoRNA in the transformed host cell. Examples of suitable expressionsystems include, for example, chromosomal, episomal and virus-derivedsystems, including, for example, vectors derived from: bacterialplasmids, bacteriophage, transposons, yeast episomes, insertionelements, yeast chromosomal elements, viruses such as baculoviruses suchas described in patent application WO2015170287, papova viruses such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, or combinations thereof, such as those derivedfrom plasmid and bacteriophage genetic elements, including cosmids andphagemids. Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained and expressed in aplasmid.

In order to express the five different recombinant proteins of the HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen simultaneously and inan equimolar way, there are several possibilities. A first possibility(1) for the HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen partof the immunogenic composition of the present invention is to build asingle vector containing all five ORFs under the control of the same orsimilar regulations elements (promoter, enhancer, splice signal,termination signal . . . ) and optionally a selection system for cellline selection. The vector could contain five expression cassettes (forinstance as described in Albers et al., J. Clin. Invest., 2015, 125(4):1603-1619; or in Cheshenko et al., Gene Ther., 2001, 8(11): 846-854), orthe five components (gH, gL, UL128, UL130 and UL131) could be fused in asingle ORF with elements triggering the proper polyprotein maturationinto the five proteins of the HCMV gH/gL/UL128/UL130/UL131 pentamericcomplex antigen (for instance self-cleavable sequences as described inSzymczak-Workman et al., Cold Spring Harb. Protoc., 2012, 2012 (2):199-204). In that second case, the equimolarity is guaranteed, assumingall cleavage occur correctly. Another possibility (2) for the HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen part of theimmunogenic composition of the present invention is to build fivevectors each expressing one component of the HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen and optionally aselection system for cell line selection. The five vectors areco-transfected in the target cell line. Any intermediate system betweenpossibility (1) and possibility (2) could also be designed to minimizethe number of vectors required and maintain each vector to a reasonablesize (less than 12 kb, for example).

Suitable expression systems include microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected or transfected with virus expressionvectors (for example, baculovirus such as described in patentapplication WO2015170287); plant cell systems transformed with virusexpression vectors (for example, cauliflower mosaic virus, CaMV; tobaccomosaic virus, TMV) or with bacterial expression vectors (for example, Tior pBR322 plasmids); or animal cell systems. Cell-free translationsystems can also be employed to produce the proteins.

Examples of suitable plant cellular genetic expression systems includethose described in U.S. Pat. Nos. 5,693,506; 5,659,122; 5,608,143 andZenk (1991). “Chasing the enzymes of secondary metabolism: Plant cellcultures as a pot of goal. Phytochemistry, 30(12), pp 3861-3863. ZessNaukUMK Tornu, 13: 253-256. In particular, all plants from whichprotoplasts can be isolated and cultured to give whole regeneratedplants can be used, so that whole plants are recovered which contain thetransferred gene. Practically all plants can be regenerated fromcultured cells or tissues, including but not limited to all majorspecies of sugar cane, sugar beet, cotton, fruit and other trees,legumes and vegetables.

HEK293 cells are suitable for transient expression of the HCMV proteinsof the pentamer complex according to the invention due to their hightransfectability by various techniques, including the calcium phosphateand polyethylenimine (PEI) methods. A useful cell line of HEK293 is onethat expresses the EBNA1 protein of EBV, such as 293-6E (Loignon, etal.: “Stable high volumetric production of glycosylated humanrecombinant IFNalpha2b in HEK293 cells.”, BMC Biotechnology 8 (2008):65). Transformed HEK293 cells have been shown to secrete high levels ofthe protein into the growth medium, thus allowing the purification ofsuch protein complexes directly from the growth medium.

CHO cells are particularly suitable mammalian hosts for industrialproduction of the HCMV proteins and in particular for industrialproduction of the HCMV gH/gL/UL128/UL130/UL131 pentameric complexantigen part of the immunogenic composition according to the invention.

Transfection can be carried out by a range of methods well known in theart including using calcium phosphate, electroporation, or by mixing acationic lipid with the material to produce liposomes which fuse withthe cell membrane and deposit their cargo inside.

Methods for purifying recombinant proteins from cell supernatant or frominclusion bodies are well known in the art. In particular the HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen part of theimmunogenic composition according to the invention may be purified bysize-exclusion chromatography.

In particular, the immunogenic composition according to the inventiondoes not comprise an HCMV virus.

In particular, the immunogenic composition according to the invention isan immunogenic composition as described herein, wherein the HCMV gB andthe HCMV gH/gL/UL128/UL130/UL131 pentameric complex are the sole HCMVantigens.

Th1-Inducing Adjuvant

“Adjuvant” as used herein, has the meaning commonly known by a manskilled in the art. In particular, it refers to agents or substancesthat modulate the immunogenicity of an antigen. “Modulate theimmunogenicity” includes enhancing the magnitude and/or duration of animmune response generated by an antigen. More specifically the adjuvantscan also be classified according to the type of immune response theyinduce in the presence of the antigen. The adjuvant(s) which can be usedin an immunogenic composition according to the invention areTh1-inducing adjuvants.

A “Th1-inducing” adjuvant can be defined as an adjuvant which enhancesthe Th1 response to an antigen or a combination of antigens.

An immune response may be broadly divided into two extreme categories,being a humoral or cell mediated immune response (traditionallycharacterized by antibody and cellular effector mechanisms of protectionrespectively). These categories of response have been termed Th1-typeresponses (cell-mediated response), and Th2-type immune responses(humoral response). In mice, Th1-type responses are often characterizedby the generation of antibodies of the IgG2a or IgG2c subtype (dependingon the mouse strain), whilst in humans these may correspond to IgG1 andIgG3 type antibodies. Th2-type immune responses are characterized by thegeneration of a broad range of immunoglobulin isotypes including in miceIgG1, IgA, and IgM. Th1-type and Th2-type immune responses are alsocharacterized by different patterns of cytokine secretion (Mosmann etal., Annual Review of Immunology, 1989, 7: 145-173; Constant et al.,Annual Review of Immunology, 1997, 15: 297-322). A Th1-type immuneresponse is associated with an increased production of IFN-γ and/or IL-2cytokines by T-lymphocytes while a Th-2 type immune response isassociated with an increased production of IL-4, IL-5, IL-6, IL-13,and/or IL-10 cytokines. The distinction of Th1 and Th2-type immuneresponses is not absolute. In reality, a subject will support an immuneresponse which is described as being predominantly Th1 or predominantlyTh2. Traditionally the best indicators of the Th1:Th2 balance of theimmune response after a vaccination or infection include directmeasurement of the production of Th1 or Th2 cytokines by T lymphocytesin vitro upon stimulation with antigen, and/or the measurement (at leastin mice) of the IgG1:IgG2a,c ratio of antigen specific antibodyresponses.

Also, in the scope of the immunogenic composition according to theinvention, an adjuvant that induces predominantly a Th1-type immuneresponse is considered as a Th1-inducing adjuvant. Preferentially theadjuvant(s) which can be used in an immunogenic composition according tothe invention induce predominantly a Th1-type immune response.

As previously mentioned, this can be determined by measurement of theIgG1:IgG2a,c ratio in mice. An increase of INF-y is an additionalindicator of predominant Th1 response. Preferably, a decreasedproduction of IL-5 is also observed.

Preferably, the Th1-inducing adjuvants which can be used in theimmunogenic composition according to the invention comprising an HCMV gBantigen and an HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigeninduce a more Th1-biased response profile than MF59 in a compositioncomprising the same HCMV gB antigen and the same HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen.

MF59 is a squalene-based oil-in-water emulsion described in patentapplication WO90/14837, U.S. Pat. Nos. 6,299,884 and 6,451,325, and inOtt et al., “MF59—Design and Evaluation of a Safe and Potent Adjuvantfor Human Vaccines” in Vaccine Design: The Subunit and Adjuvant Approach(Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York, 1995, pp.277-296).

In particular, the Th1-inducing adjuvants which can be used in theimmunogenic composition according to the invention comprising an HCMV gBantigen and an HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigeninduce a lower IgG1:IgG2a,c ratio in mice than MF59 in a compositioncomprising the same HCMV gB antigen and the same HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen.

More particularly, the Th1-inducing adjuvants which can be used in theimmunogenic composition according to the invention comprising an HCMV gBantigen and an HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigeninduce a higher INF-y level in mice than MF59 in a compositioncomprising the same HCMV gB antigen and the same HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen.

Even more particularly, the Th1-inducing adjuvants which can be used inthe immunogenic composition according to the invention comprising anHCMV gB antigen and an HCMV gH/gL/UL128/UL130/UL131 pentameric complexantigen induce a lower IL-5 level in mice than MF59 in a compositioncomprising the same HCMV gB antigen and the same HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen.

Still even more particularly, the Th1-inducing adjuvants which can beused in the immunogenic composition according to the inventioncomprising an HCMV gB antigen and an HCMV gH/gL/UL128/UL130/UL131pentameric complex antigen induce a lower IgG1:IgG2a,c ratio and ahigher INF-y level in mice than MF59 in a composition comprising thesame HCMV gB antigen and the same HCMV gH/gL/UL128/UL130/UL131pentameric complex antigen.

In particular, the Th1-inducing adjuvants which can be used in theimmunogenic composition according to the invention comprising an HCMV gBantigen and an HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigeninduce a lower IgG1:IgG2a,c ratio and a lower IL-5 level in mice thanMF59 in a composition comprising the same HCMV gB antigen and the sameHCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen.

More particularly, the Th1-inducing adjuvants which can be used in theimmunogenic composition according to the invention comprising an HCMV gBantigen and an HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigeninduce a lower IgG1:IgG2a,c ratio, a higher INF-y level and a lower IL-5level in mice than MF59 in a composition comprising the same HCMV gBantigen and the same HCMV gH/gL/UL128/UL130/UL131 pentameric complexantigen.

In particular, the immunogenic composition according to the invention isan immunogenic composition comprising:

-   -   an HCMV gB antigen;    -   an HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen; and    -   a Th1-inducing adjuvant,        wherein said Th1-inducing adjuvant induces in mice a lower        IgG1:IgG2a,c ratio, and/or a higher INF-y level, and/or a lower        IL-5 level than MF59 in a composition comprising the same HCMV        gB antigen and the same HCMV gH/gL/UL128/UL130/UL131 pentameric        complex antigen.

In particular, Th1-inducing adjuvant according to the inventioncomprises:

-   -   a TLR-4 agonist; or    -   a linear or branched polyacrylic acid polymer salt with a weight        average molecular weight Mw in the range of 350 to 650 kDa.

In particular, an immunogenic composition according to the invention isan immunogenic composition comprising:

-   -   an HCMV gB antigen;    -   an HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen; and    -   a Th1-inducing adjuvant,        wherein said Th1-inducing adjuvant comprises:    -   a TLR-4 agonist selected from the group consisting of a        lipopolysaccharide, a monophosphoryl lipid A (MPL), a        3-de-O-acylated monophosphoryl lipid A (3D-MPL), a        glucopyranosyl lipid adjuvant (GLA), a second-generation lipid        adjuvant (SLA), a phospholipid dimer connected by a        noncarbohydrate backbone and an aminoalkyl glucosaminide        phosphate, or a derivative thereof; or    -   a polyacrylic acid polymer salt with a weight average molecular        weight Mw in the range of 350 to 650 kDa.

In one embodiment, said Th1-inducing adjuvant comprises a TLR-4 agonist.

A TLR (toll-like receptor) agonist is understood to mean a natural TLRligand, a TLR ligand mimic, a synthetic or chemical TLR ligand, a cellor particle including a pathogen associated molecular pattern, amicrobial pathogen, a bacterium, a virus and viral-like particle.

TLR4 (toll-like receptor type 4) is a receptor expressed byantigen-presenting cells of the immune system; it is involved in earlydefense mechanisms against gram-bacterial infections. Thelipopolysaccharide (LPS) of gram-bacteria is the natural ligand forTLR4; it activates the receptor, which triggers a cascade of biochemicalevents, in particular the activation of Nf-Kappa B transcription factor,and the production of pro-inflammatory cytokines. The ability of acompound to stimulate the TLR4 pathway can be evaluated by methods knownby those skilled in the art, as described for instance in the Journal ofBiological Chemistry, (2001), vol 276(3), page 1873-1880.

Examples of TLR4 agonists include monophosphoryl lipid A (MPL), or aderivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A(3D-MPL) as described in GB2211502 or in U.S. Pat. No. 4,912,094, or aderivative thereof, Phosphorylated hexaacyl disaccharide also calledglucopyranosyl lipid adjuvant or GLA (CAS Number 1246298-63-4) or aderivative thereof, second-generation Lipid Adjuvant (SLA) such asdescribed in Carter et al., Clin. Transl. Immunology, 2016, 5(11):e108or in EP2437753 or U.S. Pat. No. 9,480,740, or a derivative thereof,aminoalkyl glucosaminide phosphates (AGPs) as described in WO 98/50399or in WO 01/034617, or a derivative thereof, in particular RC529described in U.S. Pat. No. 6,113,918, or a derivative thereof, andchemical compounds or a phospholipid dimer (homodimer or heterodimer)connected by a noncarbohydrate backbone as described in US 2003/0153532or in US 2005/0164988, or a derivative thereof, in particular thecompounds identified and exemplified in US 2003/0153532 under thefollowing names: ER803022 (CAS number: 287180-56-7), ER803058 (CASnumber: 287180-57-8), ER803732 (CAS number: 287106-29-0), ER803789 (CASnumber: 287180-61-4), ER804053 (CAS number: 287180-62-5), ER804057 (CASnumber: 287180-63-6), ER804058 (CAS number: 287180-65-8), ER804059 (CASnumber: 287180-64-7), ER 8044442 (CAS number: 287180-78-3), ER 804764(CAS number: 287180-87-4), ER111232 (CAS number: 287180-48-7), ER112022(CAS number: 287180-46-5), ER112048 (CAS number: 287106-02-9), ER112065(CAS number: 287180-49-8), ER112066 (CAS number: 287180-50-1), ER113651(CAS number: 287180-51-2), ER118989 (CAS number: 287180-52-3), ER119327(CAS number: 287180-54-5) and ER119328 (CAS number: 287180-55-6), or aderivative thereof. These compounds have generally one or severalasymmetric carbons. When these compounds have one or several asymmetriccarbons, they can be used as a mixture of optical isomers or under theform of a specific isomer.

In particular, said TLR-4 agonist is chosen from a phospholipid dimerconnected by a noncarbohydrate backbone and a GLA TLR-4 agonist.

In particular, the TLR4 agonist is a phospholipid dimer connected by anoncarbohydrate backbone.

In particular, the TLR4 agonist is a chemical compound or a phospholipiddimer connected by a noncarbohydrate backbone of formula I, II, III orIV:

Compound or phospholipid dimer connected by a noncarbohydrate backboneof formula I

Compound or phospholipid dimer connected by a noncarbohydrate backboneof formula II

Compound or phospholipid dimer connected by a noncarbohydrate backboneof formula III

Compound or phospholipid dimer connected by a noncarbohydrate backboneof formula IV

in which, for each of formula I, II, III or IV, R¹ is selected from thegroup consisting of:

-   -   a) C(O);    -   b) C(O)—(C₁-C₁₄ alkyl)-C(O), in which said C₁-C₁₄ alkyl is        optionally substituted with a hydroxyl, a C₁-C₅ alkoxy, a C₁-C₅        alkylenedioxy, a (C₁-C₅ alkyl)amino or a (C₁-C₅ alkyl)aryl, in        which said aryl moiety of said (C₁-C₅ alkyl)aryl is optionally        substituted with a C₁-C₅ alkoxy, a (C₁-C₅ alkyl)amino, a (C₁-C₅        alkoxy)amino, a (C₁-C₅ alkyl)-amino(C₁-C₅ alkoxy), —O—(C₁-C₅        alkyl)amino (C₁-C₅ alkoxy), —O—(C₁-C₅ alkyl)amino-C(O)—(C₁-C₅        alkyl)-C(O)OH, or —O—(C₁-C₅ alkyl)amino-C(O)—(C₁-C₅        alkyl)-C(O)—(C₁-C₅)alkyl;    -   c) an alkyl comprising a C₂-C₁₅ linear or branched chain,        optionally substituted with a hydroxyl or an alkoxy; and    -   d) —C(O)—(C₆-C₁₂ arylene)-C(O)— in which said arylene is        optionally substituted with a hydroxyl, a halogen, a nitro or an        amino;    -   a and b are independently 0, 1, 2, 3 or 4;    -   d, d′, d″, e, e′ and e″ are independently 0, 1, 2, 3 or 4;    -   X¹, X², Y¹ and Y² are independently selected from the group        consisting of null, an oxygen, NH and N (C(O)(C₁-C₄ alkyl)), and        N(C₁-C₄ alkyl);    -   W¹ and W² are independently selected from the group consisting        of a carbonyl, a methylene, a sulfone and a sulfoxide;    -   R² and R⁵ are independently selected from the group consisting        of:    -   a) a C₂ to C₂₀ straight chain or branched chain alkyl, which is        optionally substituted with an oxo, a hydroxyl or an alkoxy;    -   b) a C₂ to C₂₀ straight chain or branched chain alkenyl or        dialkenyl, which is optionally substituted with an oxo, a        hydroxyl or an alkoxy;    -   c) a C₂ to C₂₀ straight chain or branched chain alkoxy, which is        optionally substituted with an oxo, a hydroxyl or an alkoxy;    -   d) NH—(C₂ to C₂₀ straight chain or branched chain alkyl), in        which said alkyl group is optionally substituted with an oxo, a        hydroxy or an alkoxy; and    -   e)

in which Z is selected from the group consisting of an O and NH, and Mand N are independently selected from the group consisting of an alkyl,an alkenyl, an alkoxy, an acyloxy, an alkylamino and an acylaminocomprising a C₂-C₂₀ linear or branched chain; R³ and R⁶ areindependently selected from the group consisting of a C₂ to C₂₀ straightchain or branched chain alkyl or alkenyl, optionally substituted with anoxo or a fluoro; R⁴ and R⁷ are independently selected from the groupconsisting of a C(O)—(C₂ to C₂₀ straight chain or branched chain alkylor alkenyl), a C₂ to C₂₀ straight chain or branched chain alkyl, a C₂ toC₂₀ straight chain or branched chain alkoxy, and a C₂ to C₂₀ straightchain or branched chain alkenyl; in which said alkyl, alkenyl or alkoxygroups can be independently and optionally substituted with a hydroxyl,a fluoro or a C₁-C₅ alkoxy; G₁, G₂, G₃ and G₄ are independently selectedfrom the group consisting of an oxygen, a methylene, an amino, a thiol,—C(O)NH—, —NHC(O)—, and —N(C(O)(C₁-C₄ alkyl))-;or G²R⁴ or G⁴R⁷ can together be a hydrogen atom or a hydroxyl;and in which, for formula III:a′ and b′ are independently 2, 3, 4, 5, 6, 7 or 8, preferably 2;Z¹ is selected from the group consisting of —OP(O)(OH)₂, —P(O)(OH)₂,—OP(O)(OR⁸)(OH) where R⁸ is a C₁-C₄ alkyl chain, —OS(O)₂OH, —S(O)₂OH,—CO₂H, —OB(OH)₂, —OH, —CH₃, —NH₂ and —NR⁹ ₃ where R⁹ is a C₁-C₄ alkylchain;

Z² is selected from the group consisting of —OP(O)(OH)₂, —P(O)(OH)₂,—OP(O)(OR¹⁰)(OH) where R¹⁰ is a C₁-C₄ alkyl chain, —OS(O)₂OH, —S(O)₂OH,—CO₂H, —OB(OH)₂, —OH, —CH₃, —NH₂ and —NR¹¹ where R¹¹ is a C₁-C₄ alkylchain;

and in which, for formula IV:

R¹² is H or a C₁-C₄ alkyl chain;

or a pharmaceutically acceptable salt of the compound or thephospholipid dimer connected by a noncarbohydrate backbone of formula I,II, III or IV.

In particular, the TLR4 agonist according to the invention is a chemicalcompound or phospholipid dimer connected by a noncarbohydrate backboneof formula I,

or a pharmaceutically acceptable salt of this compound or phospholipiddimer connected by a noncarbohydrate backbone.

Preferably,

R¹ is C(O) or C(O)—(CH₂)n-C(O), n being 1, 2, 3 or 4,

a, b, d, d′, d″, e, e′ and e″ are independently 1 or 2,

X1, X2, Y1 and Y2 are NH,

W1 and W2 are C(O),

R² and R⁵ are independently selected from the group consisting of aC₁₀-C₁₅ straight chain alkyl optionally substituted with an oxo, anNH—(C₁₀-C₁₅ straight chain alkyl), and

in which M and N are independently a C2 to C20 straight chain alkyl oralkenyl,

R3 and R6 are C5-C10 straight chain alkyls,

R4 and R7 are selected from the group consisting of a hydrogen,C(O)—(C8-C12 straight chain alkyl) or C(O) (C8 C12 straight chainalkenyl),

G1 and G3 are an oxygen or —NH(CO)—,

G2 and G4 are an oxygen.

In particular, the TLR4 agonist according to the invention is asymmetric phospholipid dimer (homodimer) connected by a noncarbohydratebackbone. More particularly, the symmetric phospholipid dimer connectedby a noncarbohydrate backbone is a dimer of a triacyl phospholipid. Moreparticularly, said TLR-4 agonist is E6020 (CAS number: 287180-63-6).

In particular, said TLR-4 agonist is GLA (CAS Number 1246298-63-4).

These TLR4 agonists can also be themselves combined with a deliverysystem such as calcium phosphate, liposomes, virosomes, ISCOMs, micro-and nanoparticles, or emulsions.

As such, in particular, TLR4 agonist according to the invention is incombination with a delivery system such as aqueous nanosuspension,calcium phosphate, liposomes, virosomes, ISCOMs, micro- andnanoparticles, or emulsions.

Such delivery systems have been previously described and are well knownby the man skilled in the art.

In particular, said TLR-4 agonist is chosen from E6020 (CAS number:287180-63-6) and a GLA (CAS Number 1246298-63-4) TLR-4 agonist.

As an example of suitable formulation of a TLR4 agonist combined with adelivery system, citation can be made of an oil-in-water emulsioncomprising as TLR4 agonist the compound ER 804057 (now called E6020)(CAS number: 287180-63-6), which is the disodium salt of the compoundhaving the following chemical formula:

The four asymmetric carbons of E6020 are all in in the R configuration(R,R,R,R). Such an emulsion can be obtained for instance bymicrofluidisation techniques as described in WO 2004/060396 or by aphase inversion temperature process (PIT process) as described in WO2007/080308.

As such, in particular, TLR4 agonist according to the invention is incombination with an oil-in-water emulsion, more particularly, asqualene-based oil-in-water emulsion.

An oil-in-water emulsion suitable for the purpose of the inventioncomprises a metabolizable oil (wherein the volume of oil represents 0.5to 20% of the total volume of the emulsion (v/v), in particular 1 to 10%(v/v) and more particularly 1 to 5% (v/v)), an aqueous solution (whereinthe volume of the aqueous solution represents 80 to 99.5% of the totalvolume (v/v), in particular 90 to 99% (v/v)) and one or severalemulsifying agent(s) (wherein the total amount of the emulsifying agentrepresents 0.001 to 5% of the total amount of the emulsion (w/w), inparticular 0.001 to 2% (w/w), and more particularly 0.01 to 2% (w/w)).The metabolizable oil is commonly one having about 6 to about 30 carbonatoms including, but not limited to, alkanes, alkenes, alkynes, andtheir corresponding acids and alcohols, the ethers and esters thereof,and mixtures thereof. The oil can be essentially any plant oil, fishoil, animal oil or synthetically prepared oil that can be metabolized bythe body of the human subject to which the emulsion compositions will beadministered and that is not substantially toxic to the subject. Themetabolizable oil can be an unsaturated hydrocarbon having from 20-40carbons, or a branched, polyunsaturated hydrocarbon having from 20-40carbon atoms, for example, terpenoids. An unsaturated terpenoid known assqualene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaeneand its saturated analog, squalane, are often preferred. Fish oils,including squalene and squalane, are readily available from commercialsources or may be obtained by methods known in the art. Another oilcommonly used is tocopherol. Where a composition includes a tocopherol,any of the α, β, γ, δ, ε or ξ tocopherols can be used but α-tocopherolsare preferred. A substantial number of suitable emulsifying agents (alsoreferred as surfactants, detergents and so forth) are used in thepharmaceutical sciences, many of which are useful in the composition ofthe emulsion of the present invention, so long as they are sufficientlynon-toxic. There are a number of emulsifying agents specificallydesigned for and commonly used in biological situations.

For example, a number of biological detergents (surfactants) are listedas such by Sigma Chemical. Such surfactants are divided into four basictypes: anionic, cationic, zwitterionic, and nonionic.

Examples of anionic detergents include alginic acid, caprylic acid,cholic acid, 1-decanesulfonic acid, deoxycholic acid, 1-dodecanesulfonicacid, N-lauroylsarcosine, and taurocholic acid.

Cationic detergents include dodecyltrimethylammonium bromide,benzalkonium chloride, benzyldimethylhexadecyl ammonium chloride,cetylpyridinium chloride, methylbenzethonium chloride, and 4-picolinedodecyl sulfate.

Examples of zwitterionic detergents include3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (commonlyabbreviated CHAPS), 3-[(cholamidopropyl)dimethylammoniol-2-hydroxy-1-propanesulfonate (commonly abbreviatedCHAPSO), N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,phosphatidylcholine and lyso-alpha-phosphatidylcholine.

Examples of nonionic detergents include decanoyl-N-methylglucamide,diethylene glycol monopentyl ether, n-dodecyl beta-D-glucopyranoside,poloxamers, ethylene oxide condensates of fatty alcohols (e.g., thosesold under the trade name Lubrol), polyoxyethylene ethers of fatty acids(particularly C12-C20 fatty acids), polyoxyethylene sorbitan fatty acidesters (e.g., sold under the trade name Tween®), and sorbitan fatty acidesters (e.g., sold under the trade name Span®).

A particularly useful group of surfactants are the sorbitan-basednon-ionic surfactants. These surfactants are typically prepared bydehydration of sorbitol to give 1,4-sorbitan, which is then reacted withone or more equivalents of a fatty acid. The fatty-acid-substitutedmoiety may be further reacted with ethylene oxide to give a second groupof surfactants.

The fatty-acid-substituted sorbitan surfactants are typically made byreacting 1,4-sorbitan with a fatty acid such as lauric acid, palmiticacid, stearic acid, oleic acid, or a similar long chain fatty acid togive the 1,4-sorbitan mono-ester, 1,4-sorbitan sesquiester or1,4-sorbitan triester. The common names for some of these surfactantsinclude, for example, sorbitan monolaurate, sorbitan monopalmitate,sorbitan monostearate, sorbitan monooleate, sorbitan sesquioleate, andsorbitan trioleate. These surfactants are commercially available underthe names SPAN® or ARLACEL®. SPAN® and ARLACEL® surfactants arelipophilic and are generally soluble or dispersible in oil. They arealso soluble in most organic solvents. In water they are generallyinsoluble but dispersible. Generally these surfactants will have ahydrophilic-lipophilic balance (HLB) number between 1.8 and 8.6. Suchsurfactants can be readily made by means known in the art or arecommercially available.

A related group of surfactants comprises polyoxyethylene sorbitanmonoesters and polyoxyethylene sorbitan triesters. These materials aretypically prepared by addition of ethylene oxide to a 1,4-sorbitanmonoester or triester. The addition of polyoxyethylene converts thelipophilic sorbitan mono- or triester surfactant into a hydrophilicsurfactant generally soluble or dispersible in water and soluble tovarying degrees in organic liquids. The TWEEN® surfactants may becombined, for example, with a related sorbitan monoester or triestersurfactant to promote emulsion stability. TWEEN® surfactants generallyhave a HLB value falling between 9.6 and 16.7. TWEEN® surfactants arecommercially available from a number of manufacturers, for example ICIAmerica's Inc., Wilmington, Del. under the registered mark ATLAS®surfactants.

Another group of non-ionic surfactants that could be used alone or inconjunction with SPAN®, ARLACEL® and/or TWEEN® surfactants are thepolyoxyethylene fatty acids made by the reaction of ethylene oxide witha long-chain fatty acid. The most commonly available surfactant of thistype is solid under the name MYRJ® and is a polyoxyethylene derivativeof stearic acid. MYRJ® surfactants are hydrophilic and soluble ordispersible in water, like TWEEN® surfactants. The MYRJ® surfactants maybe blended, for example, with TWEEN® surfactants or with TWEEN®/SPAN® orwith ARLACEL® surfactant mixtures for use in forming emulsions. MYRJ®surfactants can be made by methods known in the art or are availablecommercially from ICI America's Inc.

Another group of polyoxyethylene based non-ionic surfactants are thepolyoxyethylene fatty acid ethers derived from lauryl, acetyl, stearyland oleyl alcohols. These materials are typically prepared as above byaddition of ethylene oxide to a fatty alcohol. The commercial name forthese surfactants is BRIJ®; BRIJ® surfactants may be hydrophilic orlipophilic depending on the size of the polyoxyethylene moiety in thesurfactant. While the preparation of these compounds is available fromthe art, they are also readily available from such commercial sources asICI America's Inc.

Other non-ionic surfactants that may be used in the practice of thisinvention are, for example: polyoxyethylenes, polyol fatty acid esters,polyoxyethylene ethers, polyoxypropylene fatty ethers, bee's waxderivatives containing polyoxyethylene, polyoxyethylene lanolinderivatives, polyoxyethylene fatty glycerides, glycerol fatty acidesters or other polyoxyethylene acid alcohols or ether derivatives oflong-chain fatty acids of 12-22 carbon atoms. Preferably, thepolyoxyethylene alkyl ether is chosen from the group consisting ofceteareth-12 (sold under the name Eumulgin® B1), ceteareth-20 (Eumulgin®B2), steareth-21 (Eumulgin® S21), ceteth-20 (Simulsol® 58 or Brij® 58),ceteth-10 (Brij® 56), steareth-10 (Brij® 76), steareth-20 (Brij® 78),oleth-10 (Brij® 96 or Brij® 97) and oleth-20 (Brij® 98 or Brij® 99). Thenumber attributed to each chemical name corresponds to the number ofethylene oxide units in the chemical formula. In a particular aspect,the polyoxyethylene alkyl ether is BRIJ® 56 or polyoxyethylene (12)cetostearyl ether, provided by the company Cognis under the nameEumulgin™ B1. Among the sorbitan ester and mannide ester basedsurfactants with a HLB less than 9 that are particularly suitable,mention may be made of the sorbitan monooleate sold under the nameDehymuls SMO™ or Span®80. Among the mannide ester-based surfactants,mention may be made of the mannide monooleate sold by the company Sigma,or by the company Seppic under the name Montanide 80™.

Two or more surfactants can be combined in the emulsion part of thecomposition of the present invention.

The aqueous solution of the O/W emulsion part of the composition of thepresent invention is buffered saline or unadulterated water. Because thecomposition of the invention is intended for parenteral administration,it is preferable to make up final buffered solutions used as vaccines sothat the tonicity, i.e., osmolality, is essentially the same as normalphysiological fluids in order to prevent post-administration swelling orrapid absorption of the composition because of differential ionconcentrations between the composition and physiological fluids. It isalso preferable to buffer the saline in order to maintain a pHcompatible with normal physiological conditions. Also, in certaininstances, it may be necessary to maintain the pH at a particular levelin order to insure the stability of the gB antigen and of the HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen if they are presentin the O/W emulsion.

Any physiologically acceptable buffer may be used herein, but phosphatebuffers are preferred. Other acceptable buffers such as acetate, tris,bicarbonate, carbonate, citrate or the like may be used as substitutesfor phosphate buffers. The pH of the aqueous component will preferablybe between 6.0 and 8.0.

The O/W emulsion part of the composition of the present invention maycomprise supplemental components that can be added at the time ofpreparing the O/W emulsion or added once the O/W emulsion is prepared.

Example can be given of AF04, a squalene-based oil-in-water (O/W)emulsion that contains E6020, which was obtained according to theprocess described in WO 2007/080308.

GLA (CAS Number 1246298-63-4) TLR-4 agonist is the compound having thefollowing chemical formula:

GLA can be purchased for instance on Avanti Polar's catalog withreference 699800 (Avanti Polar Lipids Inc., Alabaster, USA).

In particular, GLA is in combination with a delivery system such ascalcium phosphate, liposomes, virosomes, ISCOMs, micro- andnanoparticles, or emulsions. Preferentially, GLA is in combination withan oil-in-water emulsion, more particularly, a squalene-basedoil-in-water emulsion.

Example can be given of GLA-SQEM, a squalene-based oil-in-water (O/W)emulsion that contains GLA.

In the Table 1 below, the quantity of the different raw materials usedto obtain 100 mL of the GLA-SQEM with a concentration of 10% squaleneare mentioned. The final emulsion contains 4% (v/v) of squalene (34mg/ml) and 100 μg/mL of GLA, in 22.5 mM of ammonium phosphate.

TABLE 1 Preparation at Mass % Quantity 10% squalene (w/v) for 100 mLReference Aqueous phase (90%): 25 mM Ammonium 87.7 mL PhosphatepH 6.1qsp 90 ml Poloxamer 188 0.09 90.0 mg P5556 from SIGMA (PluronicF68)Glycerol 2.25 2.25 g 24388238 Normapur from VWR Prolabo Oil phase (10%)Squalene 10% v/v 8.56 10.0 mL 1208076 from SOPHIM, (85.6mg/ml)redistilled to remove peroxides Dimyristoyl- 1.9 1.90 g 850345P fromAvanti phosphatidyl- Polar Lipids choline (DMPC) GLA 0.025 25.0 mg699800P from Avanti Polar Lipids

The oil phase is prepared by sonication in a bath at 55-60° C. Then, theaqueous phase is added upon the oil phase (weighing). A pre-emulsion isobtained after homogenization on Ultra Turrax T25 (IKA) at 9500 r/min,during 2 cycles of 30 secondes. Then, microfluidization is done onEmulsiflex C3 during 20 passages at an air pressure of 55 psi(homogenization pressure between 1450 and 1600 bars). The emulsion isthen diluted 2.5 fold in a 25 mM Ammonium Phosphate buffer, to obtainthe final GLA-SQEM emulsion at 4% squalene. The emulsion is sterilefiltered at about 40° C. with a 10 ml syringe and an Acrodisc 0.8-0.2 μmSupor Membrane filter (PALL n° PN4187).

Other suitable adjuvants comprising a TLR4 agonist are AS01, whichcomprises 3D-MPL and QS21 in a liposomal formulation or AS02, whichcomprises 3D-MPL and QS21 formulated in an oil-in-water emulsion (Garçonet al., Exp. Rev. of Vaccines, 2007, 6(5):723-739, EP0671948).

In one embodiment, said Th1-inducing adjuvant comprises a linear orbranched polyacrylic acid polymer salt with a weight average molecularweight Mw in the range of 350 to 650 kDa.

Said polymer is a linear or a branched polyacrylic acid polymer, but itis not a cross-linked polymer.

By “polyacrylic acid polymer”, is meant a polymer which is exclusivelycomposed of acrylic acid units. So, in the form of a salt, saidpolyacrylic acid polymer salt is exclusively composed of unitscorresponding to a salt of acrylic acid or is exclusively composed ofunits corresponding to the free acid form of acrylic acid and of unitscorresponding to a salt of acrylic acid.

A linear or a branched polyacrylic acid polymer is obtained bypolymerization of only acrylic acid as monomer. The polymerization is,most of the time, carried out by radical polymerization, using anoxidizing agent as initiator or catalyst. The most used oxidizing agentsare persulfate (peroxydisulfate), for instance sodium or potassiumpersulfate. Branched polyacrylic acid polymers are, for instance,described in Macromolecules 2011, 44, 5928-5936. When the polymeraccording to the invention is linear, its Mark Houwink slope is higheror equal to 0.7 (Yan J. K., Pei J. J., Ma H. L., Wang Z. B. 2015.Effects of ultrasound on molecular properties, structure, chainconformation and degradation kinetics of carboxylic curdlan. Carb.Polymers. 121, 64-70).

The polyacrylic acid polymer salt can be in a solid form (precipitate orpowder) or preferably in a liquid formulation. A liquid formulation willinclude the polyacrylic acid polymer salt and an aqueous solution.Preferably, such a formulation has a pH in the range of 5.5 to 8.0. ThispH can be obtained by incorporation of a base, like NaOH, in the aqueoussolution. The aqueous solution can be a buffered aqueous solution,obtained with a buffer such as a phosphate buffer, a TRIS(2-amino-2-hydroxymethyl-1,3-propanediol), Hepes (acide4-(2-hydroxyethyl)-1-piperazine ethane sulfonique), histidine or citratebuffer. The liquid formulation may also comprise one or severaladditional salts, such as NaCl.

In particular, said linear or branched polyacrylic acid polymer salt isexclusively composed of units corresponding to a salt of acrylic acid oris exclusively composed of units corresponding to the free acid form ofacrylic acid and of units corresponding to a salt of acrylic acid.

Advantageously, said polyacrylic acid polymer salt comprises less than0.005%, preferably less than 0.001%, w/w of oxidizing agents, based onthe total dry weight of said polyacrylic acid polymer salt and/orcomprises less than 0.005%, preferably less than 0.001%, w/w ofpersulfates, based on the total dry weight of said polyacrylic acidpolymer salt.

In a more particular embodiment, said polyacrylic acid polymer is a saltwith Na+.

In particular embodiments, said polyacrylic acid polymer salt has apolydispersity index below or equal to about 4, preferably below orequal to about 2.5.

In particular embodiments, said polyacrylic acid polymer salt has aweight average molecular weight Mw in the range of 380 to 620 kDa and apolydispersity index below or equal to 4; or has a weight averagemolecular weight Mw in the range of 400 to 600 kDa and a polydispersityindex below or equal to 4; or has a weight average molecular weight Mwin the range of 380 to 620 kDa and a polydispersity index below or equalto 2.5; or has a weight average molecular weight Mw in the range of 400to 600 kDa and a polydispersity index below or equal to 2.

Advantageously, said polyacrylic acid polymer salt comprises less than0.005% w/w of acrylic acid monomer in free acid form or salt form, basedon the total dry weight of said polyacrylic acid polymer salt.

According to advantageous embodiments, said polyacrylic acid polymersalt is in a liquid formulation which has a pH in the range of 5.5 to8.0.

According to advantageous embodiments, said polyacrylic acid polymersalt is in a buffered aqueous solution, in particular with a phosphatebuffer, or a TRIS, Hepes, histidine or citrate buffer.

According to advantageous embodiments, said polyacrylic acid polymersalt is diafiltered and sterilized.

When, the polyacrylic acid polymer salt or the liquid formulation of thepolyacrylic acid polymer salt is diafiltered, the sterilization occursafter the diafiltration.

According to the invention, the weight average molecular weight Mw isobtained by size exclusion chromatography. Advantageously, threedetectors will be used after the size exclusion chromatography column: aright angle light scattering detector, a refractive index detector and afour-capillary differential viscometer. The do/dc used for thedetermination of Mw is preferably determined using the refractive indexdetector with a panel of polyacrylic acid polymers of knownconcentration. The content of persulfate and the content of acrylic acidmonomer in free acid form or salt form can be determined by HighPerformance Anion Exchange Chromatography with conductimetric detection.

A process for the preparation of such polymer comprises for example thefollowing successive steps:

-   -   a) having a solution of a polyacrylic acid polymer,    -   b) purifying the solution of the polyacrylic acid polymer, in        order to eliminate impurities,    -   and    -   c) sterilizing the purified solution of the polyacrylic acid        polymer.

A process for the storage of a solution of such polymer salt comprisesfor examples the above mentioned preparation process, followed by astorage step of the obtained polymer, in solution.

In particular, said linear or branched polyacrylic acid polymer saltwith a weight average molecular weight Mw in the range of 350 to 650 kDais PAA225000.

Product named PAA225000 (Ref. 18613, sodium salt) can be obtained fromPolysciences Europe (Eppelheim, Germany) in the form of a concentratedsolution. It can be diluted with water to obtain a concentration of 20mg/ml, and maintained under agitation at room temperature during 12hours. The pH can be adjusted to 7.55 with HCl and the solution can bedialyzed at room temperature against 150 mM NaCl aqueous solution (3consecutive baths) by using 2 kDa cutoff dialysis cassettes (ThermoFischer Scientific, Courtaboeuf, France). The solution can then befiltered through a 0.22 μm PVDF membrane, for sterilization. TheMolecular Weight of the polymer salt can then be measured and be 488 550Da. Its Mn can be 129 070 Da and its IP 3.8.

The polymer can then be stored at +4° C., as a solution comprising 20mg/ml of polymer in 150 mM NaCl aqueous solution. This solution can thenbe mixed with PBS 1C concentrated 10 times with sterile water, in orderto get a saline solution comprising 2 mg/ml of polymer salt.

Any other Th1-inducing adjuvant may be used in the composition of theinvention. As examples of adjuvants known to induce predominantly aTh1-type immune response, the following one can be cited: adjuvants orcombinations of adjuvants comprising a saponin such as the onesdescribed in WO8809336 or in U.S. Pat. No. 5,057,540, in particular QS21and its synthetic or semi-synthetic analogues, a TLR3 agonist such asPolyl:C and derivatives thereof, a TLR5 agonist such as flagellin andderivatives thereof, a TLR7 agonist or a TLR7/8 agonist such asimidazoquinoline and derivatives thereof such as the ones described inEP1318835, a TLR8 agonist such as motolimod also known as VTX-2337 (asdescribed in Lu et al., Clin Cancer Res, 2012, 18(2):499-509) andderivatives thereof or TLR8 agonists developed by Dynavax, a TLR9agonist such as CpG oligodeoxynucleotides and derivatives thereof suchas described in Vollmer et al., Expert Opin. Biol. Ther., 2005,5(5):673-682, in particular ISS1018 or CpG 7909, a RIG-I-Like receptor(RLR) agonist such as RIG-I agonist, in particular 5′ triphosphate RNAor small molecular weight agonists from Kineta, or a stimulator ofinterferon genes (STING) agonist, in particular cyclic dinucleotides(e.g. c-di-AMP, c-di-GMP, c-di-GAMP), apoly[di(carboxylatophenoxy)phosphazene] (PCPP) as described in Payne etal., Dev Biol Stand. 1998, 92:79-87, a poly[di(sodiumcarboxylatoethylphenoxy)]phosphazene (PECP) as described in Dar A etal., Vet Immunol Immunopathol., 2012, 146(3-4):289-95, or a Carbopol.

These Th1-inducing adjuvants can be combined with a delivery system suchas aqueous nanosuspension, calcium phosphate, liposomes, virosomes,ISCOMs, micro- and nanoparticles, emulsions.

The adjuvant and the antigens of the immunogenic composition accordingto the invention can be formulated with any pharmaceutically acceptablevehicle. In the context of the invention, the expression“pharmaceutically acceptable vehicle” refers to a vehicle that isphysiologically acceptable for administration to a human being, whileretaining the physiological activity of the immunogenic compositionaccording to the invention, i.e. its ability to induce an immuneresponse. One exemplary pharmaceutically acceptable vehicle is aphysiological saline buffer. Other physiologically acceptable vehiclesare known to those skilled in the art and are described, for instance,in Remington's Pharmaceutical Sciences (18th edition), ed. A. Gennaro,1990, Mack Publishing Company, Easton, Pa. An immunogenic composition asdescribed herein may optionally contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, human serumalbumin, essential amino acids, nonessential amino acids, L-argininehydrochlorate, saccharose, D-trehalose dehydrate, sorbitol, tris(hydroxymethyl) aminomethane and/or urea. In addition, the vaccinecomposition may optionally comprise pharmaceutically acceptableadditives including, for example, diluents, binders, stabilizers, andpreservatives.

The pH of the immunogenic composition is usually between 5.5 and 8, andmore preferably between 6.5 and 7.5 (e.g. about 7). Stable pH may bemaintained by the use of a buffer e.g. a Tris buffer, a citrate buffer,phosphate buffer, a Hepes buffer, or a histidine buffer. Thus, theimmunogenic composition generally includes a buffer. Immunogeniccompositions may be isotonic with respect to humans. The immunogeniccomposition may also comprise one or several additional salts, such asNaCl.

The immunogenic compositions may be sterilized by conventionalsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged and stored in liquid form orlyophilized, the lyophilized preparation being reconstituted with asterile aqueous carrier prior to administration. In a preferredembodiment, the immunogenic compositions are packaged and stored asmicropellets via a prilling process as described in WO2009109550. Eachmicropellet may comprise the gB antigen, the gH/gL/UL128/UL130/UL131pentameric complex antigen and the Th1-inducing adjuvant optionally withthe oil-in-water emulsion. Alternatively, the gB antigen, thegH/gL/UL128/UL130/UL131 pentameric complex antigen and the Th1-inducingadjuvant optionally with the oil-in-water emulsion may be comprisedalone or in any combination in different micropellets that can be mixedbefore or after aqueous reconstitution to obtain the composition of theinvention.

The adjuvant and the antigens part of the immunogenic compositionaccording to the invention are usually mixed together if there is noincompatibility between the products or alternatively the adjuvant canbe extemporaneously added just prior to administration to a subject.

In one embodiment, the immunogenic composition of the invention isprepared as a ready-to-use mix of the HCMV gB antigen, the HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen and the Th1-inducingadjuvant.

In another embodiment, the immunogenic composition of the invention isprepared extemporaneously, just before administration to the humansubjects. Thus, the invention provides kits including the variouscomponents ready for mixing. The kit allows the HCMV gB antigen, theHCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen, theTh1-inducing adjuvant and optionally the oil-in-water emulsion to bekept separately until the time of use.

These components are physically separated from each other within thekit, and this separation can be achieved in various ways. For instance,they may be in separate containers, such as vials. In some arrangements,all the components are kept separately until the time of use.Preferably, the gB antigen and the HCMV gH/gL/UL128/UL130/UL131pentameric complex antigen are in the same container and theTh1-inducing adjuvant and optionally the oil-in-water emulsion is (are)in another container. The contents of the vials can then be mixed, e.g.,by removing the content of one vial and adding it to the other vial, orby separately removing the contents of all the vials and mixing them ina new container. In one example, one or more of the kit components is(are) in syringe(s) and the other in container(s) such as a vial. Thesyringe can be used (e.g., with a needle) to insert its contents intoanother container for mixing, and the mixture can then be withdrawn intothe syringe. The mixed contents of the syringe can then be administeredto a patient, typically through a new sterile needle. In anotherarrangement, the kit components are held together but separately in thesame syringe. When the syringe is actuated (e.g., during administrationto a patient) the contents of the chambers are mixed. This arrangementavoids the need for a separate mixing step at the time of use. The kitcomponents will generally be in aqueous form. In some arrangements, oneor more component(s) is (are) in dry form (e.g., in a lyophilized formor as micropellets), with the other component(s) being in aqueous form.The components can be mixed in order to reactivate the dry component andgive an aqueous composition for administration to a patient. One or morelyophilized component(s) can be located within a vial or in a syringe.Dried components may include stabilizers such as mannitol, sucrose, ordodecyl maltoside, as well as mixtures thereof e.g. lactose/sucrosemixtures, sucrose/mannitol mixtures, etc. In some arrangements, all thecomponents are in dry form (e.g., in a lyophilized form or asmicropellets), held in the same recipient or separately in severalrecipients and the kit contains another recipient containing an aqueoussolution for the reconstitution of the vaccine.

Accordingly, the invention provides a kit comprising: (i) a first kitcomponent comprising an HCMV gB antigen and an HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen and (ii) a second kitcomponent comprising a Th1-inducing adjuvant and optionally comprisingoil-in-water emulsion; and the use of such kit for preventing HCMVinfection.

In a preferred embodiment, the immunogenic composition of the inventionis available in one vial/syringe as a ready-to-use mix of the HCMV gBantigen, the HCMV gH/gL/UL128/UL130/UL131 pentameric complex antigen andthe Th1-inducing adjuvant.

The immunogenic composition, according to the invention can beadministered via any suitable route, such as by mucosal administration(e.g. intranasal or sublingual), parenteral administration (e.g.intramuscular, subcutaneous, transcutaneous, or intradermal route), ororal administration. As appreciated by the man skilled in the art, avaccine of the present invention is suitably formulated to be compatiblewith the intended route of administration.

The immunogenic composition according to the invention can beadministered alone or with suitable pharmaceutical carriers, and can bein solid or liquid form such as, tablets, capsules, powders, solutions,suspensions, or emulsions.

For use as aerosols, the immunogenic composition according to theinvention in solution or suspension may be packaged in a pressurizedaerosol container together with suitable propellants, for example,hydrocarbon propellants like propane, butane, or isobutane withconventional adjuvants. The materials of the present invention also maybe administered in a non-pressurized form such as in a nebulizer oratomizer.

Uses

As previously mentioned, the invention also relates to an immunogeniccomposition as described herein for use as an HCMV vaccine.

In particular, the HCMV vaccine according to the invention is a subunitvaccine.

It further relates to a method of prevention of HCMV infection in apatient in need thereof, comprising the administration of animmunologically effective amount of the immunogenic compositionaccording to the invention.

“HCMV” is used as described previously and an HCMV infection can inparticular relate to a maternal HCMV infection during pregnancy or acongenital infection.

In particular, said vaccine/immunogenic composition increasesneutralizing antibody levels and/or persistence. More particularly, saidvaccine/immunogenic composition comprising an HCMV gB antigen, an HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen and a Th1-inducingadjuvant induces higher neutralizing antibody levels and/or persistencethan a vaccine/immunogenic composition comprising the same HCMV gBantigen, the same HCMV gH/gL/UL128/UL130/UL131 pentameric complexantigen and MF59 adjuvant.

By “vaccine” as used herein is meant an immunogenic composition which isadministered to induce an immune response that will protect or treat asubject from illness, in particular due to that agent. The vaccine ofthe present invention is intended for use as a preventive (prophylactic)vaccine, for administration to the subject prior to infection, with theintent to prevent initial (and/or recurrent) infection. In theparticular case of congenital HCMV infection, the present invention isintended for use as a preventive vaccine for adolescent girls and womenof child bearing age before pregnancy in order to prevent the verticalHCMV transmission from mother to fetus or infant.

An immunogenic composition according to the invention comprises animmunologically effective amount of the antigens and adjuvants describedherein. An “immunologically effective amount” is an amount which, whenadministered to a subject, is effective for eliciting an immune responseagainst the antigen used. This amount can vary depending on the healthand physical condition of the subject to be treated, their age, thecapacity of the subject's immune system to produce antibodies, thedegree of protection desired, the formulation of the vaccine, thetreating doctor's assessment of the medical situation. This amount canbe determined by routine methods by the man skilled in the art.

Subject” has mentioned herein is used in a similar way as for “patient”and refers to a human, in particular women of childbearing age (16-45y)and adolescent girls (11-15 years) whatever their CMV serostatus, aswell as a men, a child, or a patient candidate for solid organ or stemcell transplantation. In particular, said patient or subject issusceptible to contract HCMV.

A “neutralizing antibody” as described herein has the meaning known by aman skilled in the art and is intended to cover an antibody thatneutralizes its target directly, for example by blocking the virus entryinto a host cell as well as by blocking the virus dissemination fromcell to cell. Neutralizing antibodies are functional antibodies that areable to protect from their target. Some illustration of the methodsavailable to determine increase of neutralizing antibody levels and/orpersistence is provided in the experimental part of the presentapplication.

The vaccine according to the invention may be administered by any routecommonly used for administering a vaccine. A regimen leading to theinduction of the expected immune response will be used. Usually, theimmunization schedule includes several administrations. The amount ofthe immunogenic composition administered is enough to produce thedesired immune response and is determined by the person skilled in theart.

A vaccine according to the present invention may be administered inmultiple doses. For example, a vaccine according to the presentinvention may be administered in one, two or three doses. When a vaccineaccording to the present invention is administered in three doses, thefirst dose and the third dose are preferably administered approximatelytwelve months apart. For instance, a vaccine of the present inventionmay be administered in a first dose, a second dose and a third dose,wherein said second dose is to be administered about one to three monthsafter said first dose and wherein said third dose is to be administeredabout six to twelve months after said first dose. Alternatively, thethree doses may be administered at zero months, at about one to twomonths (e.g. at about one-and-a-half months) and at about six months.

A vaccine according to the present invention may be administered in twodoses. Preferably, the first dose and the second dose are administeredapproximately about one, three, six, eight or nine months apart.

A vaccine according to the present invention may be administered in asingle dose.

Optionally, booster administrations of a vaccine according to thepresent invention may be used, for example between six months and tenyears, for example six months, one year, three years, five years or tenyears after initial immunization (i.e. after administration of the lastdose scheduled in the initial immunization regimen).

All references cited herein, including journal articles or abstracts,published patent applications, issued patents or any other references,are entirely incorporated by reference herein, including all data,tables, figures and text presented in the cited references.

In the scope of the present invention, it has to be understood that “animmunogenic composition for use” is equivalent to “the use of aimmunogenic composition” and in particular that “a immunogeniccomposition for use as a vaccine” is equivalent to “the use of aimmunogenic composition as a vaccine” and to “the use of a immunogeniccomposition for the manufacture of a medicament intended to be used asvaccine”.

The invention will be further illustrated by the following figures andexamples.

FIGURES

FIG. 1 : Study schedule

FIG. 2 : Kinetic of the neutralizing antibody titers specific to HCMVBAD-rUL131-Y4 GFP strain measured by seroneutralization assay onepithelial cells with (panel A) or without complement (panel B) in seracollected from day 20 to day 257 from mice immunized at days 0, 20 and227 with 2 μg of CMV-gB and pentamer without or with differentadjuvants.

FIG. 3 : Neutralizing antibody titers specific to HCMV BAD-rUL131-Y4 GFPstrain measured by seroneutralization assay on epithelial cells with(panel A) or without complement (panel B) and on fibroblasts with (panelC) or without complement (panel D) in sera collected at day 34 from miceimmunized at days 0 and 20 with 2 μg of CMV-gB and pentamer without orwith different adjuvants.

FIG. 4 : Neutralizing antibody titers specific to HCMV BAD-rUL131-Y4 GFPstrain measured by seroneutralization assay on epithelial cells with(panel A) or without complement (panel B) and on fibroblasts with (panelC) or without complement (panel D) in sera collected at day 208 frommice immunized at days 0 and 20 with 2 μg of CMV-gB and pentamer withoutor with different adjuvants.

FIG. 5 : Neutralizing antibody titers specific to HCMV BAD-rUL131-Y4 GFPstrain measured by seroneutralization assay on epithelial cells with(panel A) or without complement (panel B) and on fibroblasts with (panelC) or without complement (panel D) in sera collected at day 257 frommice immunized at days 0, 20 and 227 with 2 μg of CMV-gB and pentamerwithout or with different adjuvants.

FIG. 6 : Anti-gB IgG1 and IgG2c antibody titers specific to CMV-gB(panel A) or to CMV-pentamer (panel B) measured by ELISA in seracollected at days 34, 208 and 257 from mice immunized at days 0, 21 and227 with 2 μg of CMV-gB and pentamer without or with differentadjuvants.

FIG. 7 : Mean IgG1/IgG2c antibody ratios calculated at day 34, 208 and257 for each group from mice immunized at 0, 21 and 227 with 2 μg ofCMV-gB and pentamer with MF59 or with different adjuvants.

FIG. 8 : IL-5 and IFN-γ cytokine secreting cells frequencies (cytokinesecreting cells/10⁶ splenocytes) upon ex-vivo stimulation withrecombinant CMV-gB (panel A) or CMV-pentamer (panel B) monitored at days34, 208 and 257 in splenocytes from mice immunized at days 0, 20 and 227with 2 μg of CMV-gB and pentamer without or with different adjuvants.

FIG. 9 : IgG1 and IgG2c percentages of antibody secreting plasmablastsspecific to either CMV-gB (panel A) or CMV-pentamer (panel B) at days34, 208 and 257 from mice immunized at days 0, 20 and 227 with 2 μg ofCMV-gB and pentamer without or with different adjuvants.

FIG. 10 : IgG1 and IgG2c percentages of antibody secreting B memorycells specific to either CMV-gB (panel A) or CMV-pentamer (panel B) atdays 34, 208 and 257 from mice immunized at days 0, 20 and 227 with 2 μgof CMV-gB and pentamer without or with different adjuvants.

FIG. 11 : Neutralizing antibody titers specific to HCMV BAD-rUL131-Y4GFP strain measured by seroneutralization assay on epithelial cellsARPE-19 or on fibroblasts MRC-5 in presence of complement in seracollected at D20 from mice immunized at D0 with different doses ofCMV-gB and CMV-gH/gL/UL128/UL130/UL131 pentamer formulated with PAAadjuvant.

FIG. 12 : Neutralizing antibody titers specific to HCMV BAD-rUL131-Y4GFP strain measured by seroneutralization assay on epithelial cellsARPE-19 in either presence (12A) or absence (12B) of complement in seracollected at D35 from mice immunized at D0 and D21 with different dosesof CMV-gB and CMV-gH/gL/UL128/UL130/UL131 pentamer formulated with PAAadjuvant.

FIG. 13 : IFN-γ cytokine-producing cells quantification in micesplenocytes upon ex-vivo stimulation with either CMV-gB,CMV-gH/gL/UL128/UL130/UL131 pentamer (panel A) or CMV-pentamer peptidepools (panel B) measured by ELISPOT assay in splenocytes collected atD35 from mice immunized at D0 and D21 with 3 μg of CMV-gB and 3 μg ofCMV-gH/gL/UL128/UL130/UL131 pentamer formulated with PAA adjuvant.

EXAMPLES

TABLE 2 List of abbreviations Acronym/AbbreviationDesignation/Description CMV Cytomegalovirus D Day ELISA Enzyme LinkedImmuno Sorbent Assay EU ELISA Unit IFNg Gamma interferon IgImmunoglobulin IL Interleukin IM Intra-muscular IP Intra-peritoneal MMonth N/A Not Applicable SN Seroneutralization W Week

Example 1

Material and Methods

Material

Product(s) Tested in the Examples

Products are described in Table 3. Female 7-weeks-old C57BL/6J mice wereimmunized by the intra-muscular (IM) route (hind leg, quadriceps) undera volume of 50 μl on D0 and D20 and D227.

TABLE 3 Product Name Concentration Source or composition Pentamer CMV 1mg/ml The Native Antigen company, Oxford UK gB (CMV) 685.5 μg/ml gBdTMobtained as described in U.S. Pat. No. 6,100,064, which is a 806 AA longpolypeptide PAA225000 8 mg/ml Polysciences Europe GmbH, Hirschberg (PAA)PAA 225000 an der Bergstrasse, Germany MF59 4% squalene Quantity per mL:Squalene 39.0 mg Polysorbate 80 4.7 mg Sorban trioleate 4.7 mg Sodiumcitrate, dehydrate 2.65 mg Citric acid, monohydrate 0.17 mg Water forinjections q.s.p. 1 mL AF04 5% squalene, Obtained according to theprocess 0.04 mg/ml E6020 described in WO 2007/080308. GLA-SQEM 4%squalene, GLA from Avanti Polar Lipids Inc., 0.1 mg/ml GLA Alabaster,USA GLA-SQEM is obtained according to the process described above in thedescription of the inventionMethods

Group Definition

Study groups are described in the following Table 4. Mice were randomlyallocated to one of the following 7 groups. Each group is differentiatedin 3 subgroups, i.e. A=>A1, A2 and A3 depending on the time-pointanalysis requiring mouse euthanasia to collect the spleens (days 34, 208and 257), as summarized in the study schedule on FIG. 1 . Thirty fivemice/groups were included as follow: 10 mice/sub-groups for sub-groups 1and 2, and 15 mice in subgroups 3 in order to compensate for possibleinter-current deaths that might happen over an eight month period. Forcontrol groups, only 5 mice/subgroup were included in subgroups A1, A2and A3 and for group B, only 5 mice/subgroup B1 and B2 were included and10 mice for subgroup B3.

TABLE 4 Sub- Groups (number Products under test of Active substanceAdjuvant Adm. Groups mouse) Name dose Name Dose route A A1 (5) PBS — — —IM (15) A2 (5) 50 μl at D0, D21 A3 (5) IM 50 μl at D0, D21, M7 B B1 (5)gB + 2 μg — — IM (20) B2 (5) Pentamer 50 μl at D0, D21 B3 (10) 2 μg IM50 μl at D0, D21, M7 C C1 (10) gB + 2 μg MF59 2% IM (35) C2 (10)Pentamer squalene 50 μl at D0, D21 C3 (15) 2 μg IM 50 μl at D0, D21, M7D D1 (10) gB + 2 μg PAA 200 μg IM (35) D2 (10) Pentamer 50 μl at D0, D21D3 (15) 2 μg IM 50 μl at D0, D21, M7 E E1 (10) gB + 2 μg AF04 1 μg IM(35) E2 (10) Pentamer 2 μg E6020, 50 μl at 2.5% D0, D21 E3 (15) squaleneIM 50 μl at D0, D21, M7 F F1 (10) gB + 2 μg GLA- 2.5 μg IM (35) F2 (10)Pentamer SQEM GLA, 50 μl at 2% D0, D21 F3 (15) 2 μg squalene IM 50 μl atD0, D21, M7

Biological Sampling and Analytical Tests

Biological Sampling

Blood samples were collected from all the animals under anesthesia. Theanesthesia was performed by Imalgene® (1.6 mg of Ketamine) and Rompun(0.32 mg of Xylazine) administered in a volume of 200 μl via theintraperitoneal route. Around 1 mL of blood was collected in vialscontaining clot activator and serum separator (BD Vacutainer SST ref367783). After a night at +4° C., blood was centrifuged at 3000 rpmduring 20 minutes and serum was collected and stored at −20° C. untilanalysis.

For cellular response assays, spleens were collected in sterileconditions and splenocytes were isolated as soon as possible afterspleen sampling.

Analytical Tests

Seroneutralization Assays

This technique is used to titrate the functional neutralizing antibodiespresent in the sera of CMV-gB+pentamer+adjuvant immunized animals. Basedon the ability of the Cytomegalovirus to infect MRC5 fibroblasts andARPE-19 cells (human epithelial cells), a serum containing specificfunctional antibodies against CMV-gB and/or CMV-pentamer can inhibit theviral infection of the cells.

Briefly, 2.5×104 MRC5 fibroblasts or ARPE-19 cells were dispensed in96-well dark plates the day before the microneutralization (MN) assay.On D0, sera were heat-inactivated at 56° C. for 30 min. Serum sampleswere serially two-fold diluted in DMEM/F12 1% FBS, starting from 1/10 to1/10240 in a 96-deep-well plate and incubated with 4.2 log FFU/ml of theBADrUL131-Y4 CMV virus strain (provided by Thomas Shenk, as described inWang et al., J. Virol., 2005, 79(16):10330-10338), titrating 4.89 or4.71 log FFU/ml on ARPE-19 or MRC5 cells, respectively) for 60 min at37° C. in a 5% CO2 cell culture incubator. The serum/virus mixtures werethen transferred onto the MRC5 or the ARPE-19 cells and incubated at 37°C. in a 5% CO₂ cell culture incubator. The incubation was performed on 3days for the MRC5 cells and on 4 days for the ARPE cells.

On D3 or D4, after removal of culture supernatant, cells were fixed with100 μl of 1% formol in PBS for 1 hour at room temperature. The plateswere then washed three times with PBS and air-dried at room temperaturebefore analysis on the Microvision fluorescent plate reader to countinfected cells in each well.

As control, two wells of cell control (without virus) and six wells withcells infected with half of the viral dilution containing the 4.2 logFFU/mL were present on each plate. The mean of these six wells definedthe threshold of seroneutralization, determined as 50% of thespecific-signal value. Neutralizing end-point titers were defined as thereciprocal of the last dilution that fell below the calculated 50%specific-signal value. Neutralizing titers (pPRNT50) were defined foreach individual serum as the last dilution that induced 50% reduction ofinfected cells, i.e. the last dilution that induced lower infected cellsthan the calculated 50% specific-signal value. Geometric meanneutralizing antibody titers were calculated for each group.

ELISA Assay

Serum IgG1 and IgG2c antibodies directed against CMV-gB antigen oragainst CMV-pentamer antigen were titrated by a robot ELISA assayaccording to the following procedure.

Dynex 96-well microplates were coated overnight at 4° C. with 1 μg/wellof CMV-gB or CMV-pentamer, in 0.05 M carbonate/bicarbonate buffer, pH9.6 (Sigma). Plates were then blocked at least 1 hour at 37° C. with 150μL/well of PBS Tween-milk (PBS pH7.1, 0.05% Tween 20, 1% (w/v) powderedskim milk (DIFCO)). All next incubations were carried out in a finalvolume of 100 μL, followed by 3 washings with PBS pH 7.1, 0.05% Tween20. Serial two-fold dilution of serum samples were performed inPBS-Tween-milk (starting from 1/1000 or 1/10000) and were added to thewells. Plates were incubated for 90 min at 37° C. After washings, goatanti-mouse IgG1 or IgG2c peroxydase conjugate antibodies (SouthernBiotech) diluted in PBS-Tween-milk at 1/2000 were added to the wells andplates were incubated for 90 min at 37° C. Plates were further washedand incubated in the dark for 30 min at 20° C. with 100 μL/well of aready-to-use Tetra Methyl Benzidine (TMB) substrate solution (TEBU). Thereaction was stopped with 100 μL/well of HCl 1M (Prolabo).

Optical density (OD) was measured at 450 nm-650 nm with a plate reader(VersaMax—Molecular Devices). The IgG1 or IgG2c antibodies titers werecalculated using the CodUnit software, for the OD value range of 0.2 to3.0 from the titration curve (reference mouse hyper immune serum put oneach plate). The IgG1 or IgG2c titer of this reference, expressed inarbitrary ELISA Units (EU) corresponds to the log 10 of the reciprocaldilution giving an OD of 1.0. The threshold of antibody detection was 10ELISA units (1.0 log 10).

All final titers were expressed in log 10 (Log).

IgG1/IgG2c ratios were calculated using the individual arithmetic valuesand the geometric mean of individual IgG1/IgG2c ratios was calculatedfor each group.

Fluorospot

The fluorescent-linked immunospot (FLUOROSPOT) is used for detecting andenumerating individual cells secreting the IFN-γ and IL-5 cytokines.

On D0, the membrane of the 96-well IPFL-bottomed microplates(Multiscreen) was pre-wetted for 1 minute with 25 μL of 35% ethanol.Ethanol was then removed and each well was washed twice with 200 μL ofPBS 1×. Microplates were then coated with a rat anti-mouse IFN-γ or ratanti-mouse IL-5 antibodies (10 μg/ml, Pharmingen) diluted at 1/100 and1/50 respectively and were incubated overnight at 4° C.

On D1, plates were washed with PBS and then blocked at least 2 h at 37°C. with RPMI 10% FBS. After plates washing, 5×10⁵ freshly isolatedsplenocytes/well were incubated overnight with the CMV-gB antigen (0.1μg/ml), CMV-pentamer (0.1 μg/ml) or concanavalin A (Con A, 2.5 μg/mL) asa positive control, in presence of murine IL-2 (10 U/ml).

On D2, the plates were washed 6 times with PBS 1×-BSA 0.1% (200μL/well). After the washing step, 100 μL/well of the biotinylatedanti-mouse IFN-γ or anti-mouse IL5 antibodies were added at 1 μg/mL inPBS1×-BSA 0.1% for 2 hours at room temperature, in the dark. The plateswere washed again 3 times with PBS 1×-BSA 0.1% (200 μL/well). Then, 100μL/well of streptavidin-PE at 1 μg/mL in PBS 1×-BSA 0.1% was incubatedfor 1 hour at room temperature, in the dark.

The plates were further washed 6 times with PBS 1×-BSA 0.1% (200μL/well). The plates were stored at 5° C.±3° C. in the dark untilreading.

Each spot, corresponding to an IFN-γ or IL5 secreting cell (IFN-γ SC orIL5 SC), was enumerated with an automatic FLUOROSPOT plate reader(Microvision). Results were expressed as number of IFN-γ or IL-5secreting cell per 10⁶ splenocytes.

IgG, IgG1 and IgG2c FLUOROSPOT Assay

The fluorescent-linked immunospot (FLUOROSPOT) is used for detecting andenumerating individual B cells secreting antibodies irrespective ofantigen specificity (IgG1, IgG2c or total IgG).

On D0, the membrane of the 96-well IPFL-bottomed microplates(Multiscreen) was pre-wetted for 1 minute with 25 μL of 35% ethanol.Ethanol was then removed and each well was washed twice with 200 μL ofPBS 1×. Microplates were then coated with CMV-gB antigen (10 μg/ml,Sanofi), CMV-pentamer (10 μg/ml, NAC) or total IgG antibody (10 μg/ml,KPL) diluted at 1/68, 1/100 and 1/100 respectively and were incubatedovernight at 4° C.

On D1, plates were washed with PBS and then blocked at least 2 h at 37°C. with RPMI 10% FBS.

After plates washing, 5×10⁵ freshly isolated splenocytes/well for CMV-gBantigen or CMV-pentamer and 2.5.10⁵ freshly isolated splenocytes/wellfor total IgG antibody were incubated 5 hours.

After 5 hours, the plates were washed 3 times with PBS 1× and stored à4° C. for the night.

On D2, the plates were washed 6 times with PBS 1×-BSA 0.1% (200μL/well). After the washing step, 100 μL/well of the anti-mouse IgG1 PEor anti-mouse IgG2c FITC or anti-mouse total IgG antibodies were addedrespectively at 4, 2 or 0.5 μg/mL in PBS1×-BSA 0.1% for 2 hours at roomtemperature, in the dark. The plates were washed again 6 times with PBS1×-BSA 0.1% (200 μL/well). The plates were stored at 5° C.±3° C. in thedark until reading.

Each spot, corresponding to an antibody secreting cell (ASC) (IgG1 ASC,IgG2c ASC or total IgG ACS), was enumerated with an automatic FLUOROSPOTplate reader (Microvision). Results were expressed as number of antibodysecreting cell per 10⁶ splenocytes.

Results

Humoral Response

Longitudinal Analysis of the Neutralizing Antibody Response on ARPE-19Epithelial Cells Between Day 20 and 257

The neutralizing activity against the BADrUL131-Y4 CMV virus strain onepithelial cells (ARPE-19) was monitored by seroneutralization assays inindividual intermediate serum samples collected monthly from all animalsfrom subgroups 3 from day 20 to day 257 (i.e. at days 20, 34, 62, 90,118, 153, 187, 226 and 257). The seroneutralization technique isdetailed in the material and methods section and raw data are shown inTables 5 a-b.

Tables 5 a-b

ARPE ARPE ARPE ARPE ARPE ARPE ARPE ARPE ARPE Group Serum D20 + C M1 + CM2 + C M3 + C M4 + C M5 + C M6 + C M7 + C M8 + C A PBS GMT 24 16 25 2923 21 17 22 16 B Pentamer: GMT 33 133 212 183 191 136 164 90 1026 2 μggB: 2 μg C Pentamer: GMT 220 3625 3516 3748 3562 2034 2973 1058 6792 2μg gB: 2 μg MF59: 2.3% squalene D Pentamer: GMT 879 15990 11598 1096210724 10266 8681 5792 37166 2 μg gB: 2 μg PAA: 200 μg E Pentamer: GMT383 12648 9288 7833 9048 5653 4494 3559 17936 2 μg gB: 2 μg AF04: 1 μgE6020, 2.5% squalene F Pentamer: GMT 976 30755 20844 15957 17068 1123111156 8505 35897 2 μg gB: 2 μg GLA-SQEM: 2.5 μg GLA, 2% squalene a-SubGroup 3-intermediary-Seroneutralization ARPE with complement

ARPE ARPE ARPE ARPE ARPE ARPE ARPE ARPE ARPE Group Serum D20 − C M1 − CM2 − C M3 − C M4 − C M5 − C M6 − C M7 + C M8 + C A PBS GMT 30 16 18 1371 22 12 12 13 B Pentamer: GMT 32 94 138 170 109 122 92 65 815 2 μg gB:2 μg C Pentamer: GMT 74 2020 1973 1928 1264 1174 1028 655 5449 2 μg gB:2 μg MF59: 2.3% squalene D Pentamer: GMT 83 3297 4890 3768 3589 32893580 2201 28657 2 μg gB: 2 μg PAA: 200 μg E Pentamer: GMT 91 3734 33543918 2728 2344 2359 1274 11910 2 μg gB: 2 μg AF04: 1 μg E6020, 2.5%squalene F Pentamer: GMT 106 8048 7446 7774 5392 4812 3459 2883 9150 2μg gB: 2 μg GLA- SQEM: 2.5 μg GLA, 2% squalene b-Sub Group3-intermediary-Seroneutralization ARPE without complement

Geometric mean titers (GMT) as well as individual neutralizing titersare depicted in FIG. 2 .

M1=D34, M2=D62, M3=D90, M4=D118, M5=D153, M6=D187, M7=D226, M8=D257.

Similar kinetics neutralizing antibody titer profiles were detected inpresence and absence of complement in the epithelial-based neutralizingassay as depicted in FIG. 2 panels A and B, respectively.

For the group administered with unadjuvanted CMV-gB and pentamer, a lowneutralizing antibody response was detected at day 20 (GMT=33 and 32,with or without complement, respectively) and then increased up to day62 to reach a plateau with GMT ranging from 90 to 212 or from 65 to 170,in presence or absence of complement, between day 62 and day 226. The3rd injection at day 226, boosted the neutralizing antibody titers asdetected at days 257 with GMT=1026 or 815, in presence or absence ofcomplement, respectively.

For all the adjuvanted groups (MF59, PAA, AF04 and GLA-SQEM),neutralizing antibody titers were detected in presence or absence ofcomplement on day 20 (i.e. 20 days after the first injection) with GMTsat 220 or 74, respectively, for group C3 administered with CMV-gB andpentamer adjuvanted with MF59 and with GMTs≥383 and ≥83 for groups D3 toF3 administered with the other adjuvant formulations. At day 34 (i.e. 14days after the 2nd injection), all the adjuvanted groups presented thepeak of the response after 2 injections with GMTs ranging from 3 625 to30 755 or from 2 020 to 8 048, in presence or absence of complement,respectively.

Over the 6 month period (between day 34 and 226), the epithelial-basedneutralizing antibody titers slightly decreased down to titers rangingfrom 1 058 to 8 505 or from 655 to 2883, in presence or absence ofcomplement, respectively. Similarly the 3rd injection at day 226,boosted the neutralizing antibody titers as detected at days 257 withGMTs ranging 6 792 (i.e. for MF59-) to 37 166 (i.e. for PAA-) or from 5449 (i.e. for MF59-) to 28 657 (i.e. for -PAA adjuvanted group), inpresence or absence of complement, respectively.

To compare the different adjuvanted groups i.e. SPA09, AF04 and GLA-SQEMto the MF59 reference, a statistical mixed model with 2 fixed factors(group and time) was performed on repeated neutralizing antibody titersbetween days 34 to 226.

With respect to the group comparison as presented in Table 6, inpresence of complement, the neutralizing antibody titers obtained inmice administered with CMV-gB and pentamer adjuvanted with MF59 were notsignificantly superior to the neutralizing antibody titers obtained inmice administered with unadjuvanted CMV-gB and pentamer whereas all theother adjuvanted groups (i.e. PAA, AF04 and GLA-SQEM) were significantlysuperior to the neutralizing antibody titers obtained in miceadministered with CMV-gB and pentamer adjuvanted with MF59 (all p-values<0.001).

In absence of complement, the neutralizing antibody titers obtained inmice administered with CMV-gB and pentamer adjuvanted with MF59 were notsignificantly superior to the neutralizing antibody titers obtained inmice administered with unadjuvanted CMV-gB and pentamer. Theneutralizing antibody titers obtained in mice administered with CMV-gBand pentamer adjuvanted with AF04 were not significantly superior to theneutralizing antibody titers obtained in mice administered with CMV-gBand pentamer adjuvanted with MF59, whereas all the other adjuvantedgroups (i.e. PAA and GLA-SQEM) were significantly superior to theneutralizing antibody titers obtained in mice administered with CMV-gBand pentamer adjuvanted with MF59 (all p_values 0.009). The neutralizingantibody titers obtained in mice administered with CMV-gB and pentameradjuvanted with AF04 were significantly superior to the neutralizingantibody titers obtained in mice administered with unadjuvanted CMV-gBand pentamer (all p-values <0.001).

TABLE 6 In presence of In absence of ARPE-19 neutralizing assayComplement Complement Comparison P-value* P-value* (B3) unadjuvant vs(C3) MF59 1.000 (NS) 1.000 (NS) (D3) PAA vs (C3) MF59 <0.001 (S) 0.009(S) x4.2 x2.4 (E3) AF04 vs (C3) MF59 <0.001 (S) 0.077 (NS) x2.8 (F3)GLA-SQEM vs (C3) MF59 <0.001 (S) <0.001 (S) x6.6 x4.0 Statisticalcomparison of the different groups within estimated repeatedneutralizing antibody titers between days 34 to 226 (Superiority test,*p-values with Dunnett adjustment, NS: not significant or S: significantsuperiority, when significant the fold increase is indicated in italic).

Detailed Neutralizing Antibody Response on Epithelial Cells (ARPE-19)and Fibroblasts (MRC-5) at Days 34 (M1), 208 (M7) and 257 (M8)

The neutralizing activity against the BADrUL131-Y4 CMV virus strain onepithelial cells (ARPE-19) and fibroblasts (MRC-5) was monitored byseroneutralization assays in individual serum samples collected from allanimals from subgroups 1, 2 and 3 at, respectively, days 34 (2 weeksafter the second immunization), 208 (7 months after the primaryvaccination series) and 257 (1 month after the booster injection at M7).The seroneutralization technique is detailed in the material and methodssection and raw data are shown in Tables 7 a-f.

Tables 7 a-f

ARPE ARPE Group Serum J34 +C J34 −C A PBS GMT 12 12 B Pentamer: 2 μg GMT46 43 gB: 2 μg C Pentamer: 2 μg GMT 4168 1934 gB: 2 μg MF59: 2.3%squalene D Pentamer: 2 μg GMT 14698 3862 gB: 2 μg PAA: 200 μg EPentamer: 2 μg GMT 7179 2919 gB: 2 μg AF04: 1 μg E6020, 2.5% squalene FPentamer: 2 μg gB: 2 μg GMT 13302 3146 GLA-SQEM: 2.5 μg GLA, 2% squalenea.- Sub Group 2- Seroneutralization ARPE D234 (D208)

ARPE ARPE Group Serum M7 +C M7 −C A PBS GMT 14 17 B Pentamer: 2 μg GMT46 51 gB: 2 μg C Pentamer: 2 μg GMT 3739 1834 gB: 2 μg MF59: 2.3%squalene D Pentamer: 2 μg GMT 4484 2230 gB: 2 μg PAA: 200 μg E Pentamer:2 μg GMT 6101 2718 gB: 2 μg AF04: 1 μg E6020, 2.5% squalene F Pentamer:2 μg GMT 7719 1758 gB: 2 μg GLA-SQEM: 2.5 μg GLA, 2% squalene b- SubGroup 2- Seroneutralization ARPE M7 (D208)

ARPE ARPE Group Serum M8 +C M8 −C A PBS GMT 16 13 B Pentamer: 2 μg GMT1026 815 gB: 2 μg C Pentamer: 2 μg GMT 6792 5449 gB: 2 μg MF59: 2.3%squalene D Pentamer: 2 μg GMT 37166 28657 gB: 2 μg PAA: 200 μg EPentamer: 2 μg GMT 17936 11910 gB: 2 μg AF04: 1 μg E6020, 2.5% squaleneF Pentamer: 2 μg GMT 8505 9150 gB: 2 μg GLA-SQEM: 2.5 μg GLA, 2%squalene c- Sub Group 3- Seroneutralization ARPE M8 (D257)

MRC5 MRC5 Group Serum D34 +C D34 −C A PBS GMT 5 5 B Pentamer: 2 μg GMT 85 gB: 2 μg C Pentamer: 2 μg GMT 636 71 gB: 2 μg MF59: 2.3% squalene DPentamer: 2 μg GMT 2699 84 gB: 2 μg PAA: 200 μg E Pentamer: 2 μg GMT1477 153 gB: 2 μg AF04: 1 μg E6020, 2.5% squalene F Pentamer: 2 μg GMT3131 146 gB: 2 μg GLA-SQEM: 2.5 μg GLA, 2% squalene d- Sub Group 1-Seroneutralization MRC5 - D34

MRC5 MRC5 Group Serum M7 +C M7 −C A PBS GMT 5 5 B Pentamer: 2 μg GMT 5 5gB: 2 μg C Pentamer: 2 μg GMT 147 27 gB: 2 μg MF59: 2.3% squalene DPentamer: 2 μg GMT 500 39 gB: 2 μg PAA: 200 μg E Pentamer: 2 μg GMT 45148 gB: 2 μg AF04: 1 μg E6020, 2.5% squalene F Pentamer: 2 μg GMT 913 14gB: 2 μg GLA-SQEM: 2.5μg GLA, 2% squalene e- Sub Group 2-Seroneutralization MRC5 M7 (D208)

MRC5 MRC5 Group Serum M8 +C M8 −C A PBS GMT 5 5 B Pentamer: 2 μg GMT 2915 gB: 2 μg C Pentamer: 2 μg GMT 695 151 gB: 2 μg MF59: 2.3% squalene DPentamer: 2 μg GMT 6645 536 gB: 2 μg PAA: 200 μg E Pentamer: 2 μg GMT1977 438 gB: 2 μg AF04: 1 μg E6020, 2.5% squalene F Pentamer: 2 μg GMT6152 427 gB: 2 μg GLA-SQEM: 2.5 μg GLA, 2% squalene f- Sub Group 3-Seroneutralization MRC5 M8 (D257)

Geometric mean titers (GMT) as well as individual neutralizing titersare depicted in FIG. 3 , FIG. 4 and FIG. 5 .

Similar neutralizing antibody profiles were observed on both epithelial-and fibroblast-based neutralizing assays, with higher neutralizingtiters monitored in the epithelial-based neutralizing assay with atleast 5-fold or 11-fold higher GMTs in presence or absence ofcomplement, respectively.

At day 34, i.e. 14 days after the 2nd injection, no or low neutralizingantibody titers were detected in mice immunized with unadjuvanted CMV-gBand pentamer (GMT 8 on MRC-5 and 46 on ARPE-19 cells, respectively). Forall the CMV-gB and pentamer adjuvanted groups, a marked adjuvant effectwas observed with a 14- up to 337-fold increase of the SN titers onMRC-5 and 44- to 319-fold increase on ARPE-19 cells, irrespective of thepresence or absence of complement, compared to the unadjuvanted group.

With respect to the neutralizing antibody titers on ARPE-19 epithelialcells in presence of complement (FIG. 3 , panel A), an adjuvant effectwith significantly higher neutralizing antibody titers than MF59 wasobserved with PAA and GLA-SQEM (at least 3- to 4.2-fold higher, test ofsuperiority, unilateral Dunnet adjustment, all p_values <0.001) but notwith AF04 (only 1.7-fold higher, p_value=0.08).

At the opposite, with respect to the neutralizing antibody titers onARPE-19 cells in absence of complement (FIG. 3 , panel B), PAA, AF04 andGLA-SQEM adjuvants slightly increased the neutralizing antibody titersas compared to MF59 (1.5- to 2-fold increase in neutralizing antibodytiters as compared to those induced by MF59) but the observeddifferences were not statistically significant (all p_values>0.091).

With respect to the neutralizing antibody titers on MRC-5 fibroblasts inpresence of complement (FIG. 3 , panel C), an adjuvant effect withsignificantly higher neutralizing antibody titers than MF59 was observedwith all the tested adjuvants PAA, AF04 and GLA-SQEM (at least 2.3- to6-fold higher, test of superiority, unilateral Dunnet adjustment, allp_values ≤0.002).

Lastly, with respect to the neutralizing antibody titers on MRC-5fibroblasts in absence of complement (FIG. 3 , panel D), theneutralizing antibody titers induced by PAA, AF04 and GLA-SQEM were notshown to be significantly higher than those obtained with MF59(p_values >0.093). At day 208 (FIG. 4 ), i.e. 7 months after the 2ndinjection, no or low neutralizing antibody titers were detected in miceimmunized with unadjuvanted CMV-gB and pentamer (GMT≤5 on MRC-5 and ≤50on ARPE-19 cells, respectively). In adjuvanted sub-groups 2, nosignificant decrease of neutralizing titers on ARPE-19 epithelial cellswas exhibited as compared to the titers detected at day 34 (in mice fromsubgroups 1), whatever the presence or absence of complement, whereassignificant decrease was evidenced in neutralizing antibody titers onMRC-5 fibroblasts (2.3- to 5.4-fold decrease in presence of complement,all p_values ≤0.016; 3- to 10-fold decrease in absence of complement,all p_values <0.001).

With respect to the neutralizing antibody titers on ARPE-19 epithelialcells in presence or absence of complement (FIG. 4 , panel A and B), nosignificant difference was detected between the tested adjuvants and theMF59 benchmark. With respect to the neutralizing antibody titers onMRC-5 fibroblasts in presence of complement (FIG. 4 , panel C), theneutralizing antibody titers induced by PAA and GLA-SQEM weresignificantly higher than those induced by MF59 (at least 3.4- to11.9-fold higher, test of superiority, unilateral Dunnet adjustment, allp_values ≤0.015).

Lastly, with respect to the neutralizing antibody titers on MRC-5fibroblasts in absence of complement (FIG. 4 , panel D), theneutralizing antibody titers induced by PAA and AF04 were significantlyhigher than those induced by MF59 (2- to 4.4-fold increase, test ofsuperiority, unilateral Dunnet adjustment, all p_values ≤0.019).

At day 257 (FIG. 5 ), i.e. 30 days after the 3rd injection, neutralizingantibody titers in mice immunized with unadjuvanted CMV-gB and pentamerincreased significantly compared to the titers detected at day 34 onARPE-19 cells (GMT=1062 or 815 on ARPE-19 cells with and withoutcomplement, respectively). At the opposite, the neutralizing antibodytiters in mice immunized with unadjuvanted CMV-gB and pentamer remainedlow on MRC-5 fibroblasts (GMT=29 or 15 on MRC-5 fibroblasts with andwithout complement, respectively).

In all adjuvanted sub-groups 3 except MF59, the neutralizing antibodytiters detected at day 257 after the 3rd injection were significantlyhigher than those detected at day 34 after the 2nd injection, whateverthe cell type and whatever the presence or absence of complement (allp_values ≤0.002).

At day 257, with respect to the adjuvant comparison to the MF59reference, all the adjuvants (i.e. PAA and AF04) except GLA-SQEM inducedhigher neutralizing antibody titers than MF59, whatever the cell typeand whatever the presence or absence of complement (test of superiority,unilateral Dunnet adjustment, all p-values ≤0.05). Regarding GLA-SQEMthe induced complement dependent neutralizing antibody titers weresignificantly higher to those induce by MF59 (5.3- or 8.9-fold higher inARPE-19 or MRC-5 cells respectively, test of superiority, unilateralDunnet adjustment, all p_values <0.001), whereas in absence ofcomplement the induced neutralizing were not significantly different,whatever the cell type.

At D208, i.e. up to 7 months after the 2^(nd) injection the compositioncomprising gB+Pentamer+AF04 or PAA or GLA-SQEM give higherneutralization antibody levels than a composition comprisinggB+Pentamer+MF59, showing a better persistence of the functionality ofthe antibodies. At D257, 1 month after the boost, the measuredneutralizing antibody increase reflects the memory response and showshigher titers for composition comprising gB+Pentamer+AF04 or PAA orGLA-SQEM than a composition comprising gB+Pentamer+MF59.

All these results show that the immunogenic composition comprisinggB+Pentamer+AF04 or PAA or GLA-SQEM give higher neutralization antibodylevels and persistence than a composition comprising gB+Pentamer+MF59.

IgG1 and IgG2c Antibody Responses

CMV gB-specific and pentamer-specific IgG1 and IgG2c antibody responseselicited by the CMV gB and pentamer antigens administrated without orwith different adjuvants were measured by ELISA in individual serumsamples collected from all animals from subgroups 1, 2 and 3 at,respectively, days 34 (2 weeks after the second immunization), 208 (7months after the primary vaccination series) and 257 (1 month after thebooster injection at M7). Mean ELISA antibody titers (log 10 EU) aredepicted in FIG. 6 . ELISA technique is detailed in the material andmethod section.

With respect to the IgG1 and IgG2c antibody responses similar profileswere obtained irrespective of the CMV-antigen specificity either gB orpentamer, whatever the analyzed time-point.

Regarding IgG1 antibody titers, all the tested adjuvants significantlyincreased the IgG1 antibody titers compared to the unadjuvanted group.No significant difference was shown for AF04 when compared to MF59,whatever the antigen and the time-point. An adjuvant effect withsignificantly lower IgG1 titers than MF59 was observed for PAA at day 34and 208 (at least 2.4-fold decrease, all p-values ≤0.045, test ofdifference, unilateral Dunnet adjustment) but not at day 257 after the3rd booster injection. Compared to the MF59 reference, GLA-SQEM inducedsignificantly lower anti-gB IgG1 titers (at least 2.5-fold decrease, allp-values ≤0.033, test of difference, unilateral Dunnet adjustment) atall the tested time-point and lower anti-pentamer IgG1 titers (at least2.7-fold decrease, all p-values ≤0.005, test of difference, unilateralDunnet adjustment) at day 208 and 257. Regarding IgG2c antibody titers,all the tested adjuvants significantly increased the IgG2c antibodytiters compared to the unadjuvanted group. An adjuvant effect withsignificantly higher IgG2c titers than MF59 was observed for all thetested adjuvants i.e. PAA, AF04 and GLA-SQEM (at-least 11-fold higher;all p-values <0.001, test of difference, unilateral Dunnet adjustment)either for IgG2c specific to gB or pentamer whatever the time-point.

ELISA IgG1/IgG2c Ratio

In order to evaluate the Th2/Th1 orientation, IgG1/IgG2c ratios werecalculated for all the adjuvanted groups and are detailed in FIG. 7 .

As shown in FIG. 7 , the IgG1/IgG2c ratios calculated for CMV-pentamerwere lower than those calculated for CMV-gB and tented to be constantwhatever the time-point. The squalene emulsion MF59 showed a Th2-biasedresponse profile with IgG1/IgG2 ratio for CMV-gB or ≥18 forCMV-pentamer, whatever the time-point. For all the other testedadjuvants, lower IgG1/IgG2c ratios than MF59 were obtained, withIgG1/IgG2c ratio specific to gB ≥7.1 and specific to pentamer 2.1 forAF04 and inferior or equal to 2.4 or 0.8 (specific to gB and pentamer,respectively) for PAA and GLA-SQEM, indicating a more Th1-biasedresponse profile than the MF59 and that AF04, PAA and GLA-SQEM areTh1-inducing adjuvants.

Cellular Response

IL5 and IFN-γ Cytokine Secreting Cells Monitored by FLUOROSPOT

The IL5 and IFN-γ secreting cell frequencies were measured by FLUOROSPOTon splenocytes collected from all animals from subgroups 1, 2 and 3 at,respectively, days 34 (2 weeks after the second immunization), 208 (7months after the primary vaccination series) and 257 (1 month after thebooster injection at M7). During the FLUOROSPOT assay, each splenocytesuspension was ex-vivo stimulated overnight with either 0.1 μg/ml ofrecombinant CMV-gB or CMV-pentamer.

The FLUOROSPOT technique is detailed in the material and methodssection.

As shown in FIG. 8 , at day 34, upon CMV-gB stimulation (panel A), no orvery low IL-5 secreting cell (SC) frequencies were detected in all thegroups (geometric mean <22 IL-5 secreting cells/10⁶ splenocytes) exceptfor the MF59 adjuvanted CMV-gB and pentamer group (geometric mean of 60IL-5 secreting cells/10⁶ splenocytes). Similarly, no or few IFN-γsecreting cell frequencies were detected in all the groups (geometricmean <20 IFN-γ secreting cells/10⁶ splenocytes).

At the opposite, high cytokine secreting cell frequencies were detectedupon CMV-pentamer stimulation (FIG. 8 , panel B). With respect to theIL-5 secreting cells, high IL-5 SC frequencies were detected in miceadministered with MF59 (444 IL-5 SC/10⁶ splenocytes). IL-5 secretionsdetected in groups administered with PAA and GLA-SQEM were significantlylower than those obtained with MF59 (p-values ≤0.002, test ofdifference, unilateral Dunnet adjustment) whereas no significantdifferences were recorded with AF04.

With respect to the IFN-γ secreting cells frequencies, all the testedadjuvants i.e. PAA, AF04 and GLA-SQEM a significant 8- up to 29-foldincrease of IFN-γ production was recorded compared to MF59 (all p-values≤0.001, test of difference, unilateral Dunnet adjustment).

At days 208, as shown in FIG. 8 , both IL-5 and IFN-γ secreting cellfrequencies were low, whatever the stimulation antigen.

At day 257, both IL-5 and IFN-γ responses upon CMV-gB and CMV-pentamerstimulation increased as compared to day 34, however the Th1/Th2profiles were conserved. With respect to the IL-5 secreting cells, highIL-5 SC frequencies were detected in mice administered with MF59 (268and 2284 IL-5 SC/10⁶ splenocytes upon CMV-gB or pentamer stimulation,respectively).

IL-5 secretions detected in groups administered with PAA, AF04 andGLA-SQEM were significantly lower than those obtained with MF59(p-values ≤0.003, test of difference, unilateral Dunnet adjustment).

With respect to the IFN-γ secreting cells frequencies, all the testedadjuvants i.e. PAA, AF04 and GLA-SQEM a significant increase of IFN-γ SCfrequencies was recorded compared to MF59 (all p-values ≤0.001, test ofdifference, unilateral Dunnet adjustment). Taking together, all thetested adjuvants induced a more Th-1 biased overall response profilethan MF59 consistent with the trend indicated by IgG1/IgG2c ratio.

Consistent with the trend indicated by IgG1/IgG2c ratio, takingtogether, all the tested adjuvants induced a Th-1 biased overallcellular response profile while MF59 induced a Th2-biased overallcellular response profile.

IgG1 and IgG2c Antibody Secreting Plasmablasts Monitored by ELISPOT

The IgG1 and IgG2c antibody secreting plasmablast frequencies weremeasured by ex-vivo FLUOROSPOT on splenocytes collected from all animalsfrom subgroups 1, 2 and 3 at, respectively, days 34 (2 weeks after thesecond immunization), 208 (7 months after the primary vaccinationseries) and 257 (1 month after the booster injection at M7). During theELISPOT assay, each splenocyte suspension was deposited in wells coatedeither with recombinant CMV-gB or CMV-pentamer to capture either IgG1 orIgG2c specific antibodies presented at the plasmablast cell surface.IgG1 and IgG2c CMV-gB and pentamer-specific Antibody Secreting cells areenumerated and reported according the total IgG secreting cells;percentage of either IgG1 or IgG2c on total IgG are calculated. TheFLUOROSPOT technique is detailed in the material and methods section.

As presented in FIG. 9 , the means of IgG1 antibody secreting cells(ASC) frequencies at day 34 were ranging between 3.8% and 20.12% withoutsignificant differences between all the tested adjuvants. Regarding theIgG2c ASC frequencies, low % were detected when mice were administeredwith CMV-gB and pentamer adjuvanted with MF59. At the opposite, CMV-gBand pentamer adjuvanted with PAA, AF04 and GLA-SQEM inducedsignificantly higher % of IgG2c ASC than MF59 (all p_values <0.001, testof difference, unilateral Dunnet adjustment), whatever the antigenspecificity either CMV-gB or CMV-pentamer.

As expected, with respect to the detected ASC at day 208, responses werelow, indicating that 6 months after the primary vaccination series, lowrates of circulating plasmablasts were detected in mouse spleens.

Thirty days after the 3rd injection (at day 257), the ASC frequencies,either IgG1 or IgG2c specific to CMV-gB or CMV-pentamer, increased ascompared to day 208. Again, the means of IgG1 ASC frequencies at day 257were ranging between 3.1% and 9% without significant differences betweenall the tested adjuvants. Regarding the IgG2c ASC frequencies, low %were detected when mice were administered with CMV-gB and pentameradjuvanted with MF59. At the opposite, CMV-gB and pentamer adjuvantedwith PAA, AF04 and GLA-SQEM induced significantly higher % of IgG2c ASCthan MF59 (all p_values <0.001, test of difference, unilateral Dunnetadjustment), whatever the antigen specificity either CMV-gB orCMV-pentamer.

IgG1 and IgG2c Antibody Secreting B Memory Cells Monitored by FLUOROSPOT

The IgG1 and IgG2c antibody secreting cells frequencies were measured byFLUOSPOT at day 34, 208 and 257 on activated and enriched B cellsplenocyte cultured for 4 days upon in vitro stimulation with IL-2 andR848. The FLUOROSPOT technique is detailed in the material and methodssection.

As presented in FIG. 10 , the means of IgG1 antibody secreting cells(ASC) frequencies at day 34 were ranging between 1.24% and 4.68% withoutsignificant differences between all the tested adjuvants and withsimilar profiles regarding the antigen specificity either CMV-gB orCMV-pentamer. Regarding the IgG2c ASC frequencies, low % were detectedwhen mice were administered with CMV-gB and pentamer adjuvanted withMF59. At the opposite, CMV-gB and pentamer adjuvanted with PAA, AF04 andGLA-SQEM induced significantly higher % of IgG2c ASC than MF59 (allp_values <0.001, test of difference, unilateral Dunnet adjustment),whatever the antigen specificity either CMV-gB or CMV-pentamer.

With respect to the detected ASC at day 208, B memory cells weredetected mainly for IgG1 ASC specific to CMV-pentamer with % rangingfrom 1.6% to 3.24% independently of the tested adjuvant. Regarding theIgG2c ASC frequencies, low % were detected when mice were administeredwith CMV-gB and pentamer adjuvanted with MF59. At the opposite, CMV-gBand pentamer adjuvanted with PAA, AF04 and GLA-SQEM inducedsignificantly higher % of IgG2c ASC than MF59 (all p_values <0.001, testof difference, unilateral Dunnet adjustment).

Thirty days after the 3rd injection (at day 257), the means of IgG1 ASCfrequencies were ranging between 1.1% and 3.75% without significantdifferences between all the tested adjuvants.

Regarding the IgG2c ASC frequencies, low % were detected when mice wereadministered with CMV-gB and pentamer adjuvanted with MF59. At theopposite, CMV-gB and pentamer adjuvanted with PAA and GLA-SQEM inducedsignificantly higher % of IgG2c ASC than MF59 (all p_values <0.001, testof difference, unilateral Dunnet adjustment), whatever the antigenspecificity either CMV-gB or CMV-pentamer.

These results show a higher memory response level with a compositioncomprising gB+pentamer+PAA or AF04 or GLA-SQEM than a compositioncomprising gB+pentamer+MF59. It is clear also that this higher memorycell frequency, which is known to be the mediator of the protectionpersistence, keeps a predominant Th1-type response profile.

Example 2

Complementary Effect of the Two Antigens

In a design of experiment study the inventors studied the combineddose-ranging effect of the two antigens in presence of PAA adjuvant. Forthat purpose 11 groups of 10 female C57/BI6J mice received byintra-muscular route on days 0 and 22 doses ranging from 0 to 5 μg ofCMV-gH/gL/UL128/UL130/UL131 pentamer with or without doses ranging from1.2 to 5 μg of CMV-gB in presence of PAA adjuvant. The antibody responsewas assessed by ELISA specific to gB and gH/gL/UL128/UL130/UL131pentamer (IgG1/IgG2c subclasses) and neutralization assays on D22 (withcomplement, on ARPE-19 epithelial cells) and D35 (with and withoutcomplement, on MRC5 fibroblasts and ARPE-19 epithelial cells). Thecellular response was assessed on D35 by IFN-y ELISPOT upon in-vitrostimulation with gB and pentamer recombinant proteins and pentamerpeptide pools.

The neutralizing activities monitored either on epithelial cells ARPE-19or fibroblasts MRC-5 presented similar profiles, with higherneutralizing titers recorded on epithelial cells than fibroblasts (2- to5-fold higher titers in ARPE-19 than MRC-5 cells). On day 20, (FIG. 11), i.e. 20 days after the 1^(st) administration, the neutralizingantibody titers inhibiting both epithelial cells and fibroblastsinfection in presence of baby rabbit complement increased depending onthe administered dose of gB and gH/gL/UL128/UL130/UL131 pentamer. Ahigher neutralizing titers increase was evidenced when the gBconcentration increased. As depicted in FIG. 11 , the radar plot isoriented according to the gB dosage rather than the pentamer dosage.Therefore, a significant linear effect of the addition of gB on the topof gH/gL/UL128/UL130/UL131 pentamer was observed for neutralizingactivities monitored on epithelial cells and on fibroblasts (p=0.014 onepithelial cells and p=0.006 on fibroblasts).

In conclusion, in presence of complement, the addition of the gB on thetop of pentamer allows to increase the SN titers on both epithelial andfibroblast cells.

On day 35, i.e. 14 days after the 2nd administration, high neutralizingantibody titers inhibiting both epithelial cells and fibroblastsinfection in presence of baby rabbit complement were detected whateverthe administered doses of either gB or gH/gL/UL128/UL130/UL131 pentamer.The detected neutralizing activities were at a plateau with nosignificant dose effect for neither the pentamer nor the gB (allp-values ≥0.240) (FIG. 12A).

On day 35, the complement independent neutralizing activities inhibitingboth epithelial cells and fibroblasts infection in absence of babyrabbit complement was also monitored.

As depicted in FIG. 12B, in absence of complement the radar plot isoriented according to the pentamer dosages rather than the gB dosages,therefore the complement independent neutralizing antibody titers highlyincreased when the gH/gL/UL128/UL130/UL131 pentamer dose increased. Nosignificant dose effect of the gB was evidenced whereas a significantlinear and square effect was evidenced for the gH/gL/UL128/UL130/UL131pentamer increasing doses (p≤009 for both the neutralizing titers onARPE-19 and MRC-5 cells).

In conclusion, in absence of complement, the addition of the pentamer onthe top of gB allows to increase the SN titers on both epithelial andfibroblast cells Thus, complementary effect of the two antigens wasevidenced by their respective effect on the neutralizing antibodyresponse quality. With respect to the analysis of the functional humoralresponses, i.e. the complement dependent and independent neutralizingantibodies, it was demonstrated that the combination of the two antigensprovided an extended mode of action for virus neutralization. CMV-gBallows increasing neutralizing antibody titers on epithelial cells andfibroblasts in presence of complement and CMV-gH/gL/UL128/UL130/UL131pentamer allows achieving complement independent neutralizing antibodyon epithelial cells and fibroblasts.

Moreover, this broadening property of the CMV-gB andCMV-gH/gL/UL128/UL130/UL131 pentamer combination was also noticed on theinduced cellular responses. As depicted in FIG. 13 panel A, specificIFN-γ cellular response was detected in splenocytes from miceadministered with CMV-gB and CMV-gH/gL/UL128/UL130/UL131 pentamerformulated with PAA adjuvant. Higher specific IFN-y cellular responsewas detected upon ex-vivo stimulation with CMV-gH/gL/UL128/UL130/UL131recombinant pentamer than CMV-gB recombinant protein. In order to definethe cellular epitopes within the CMV-gH/gL/UL128/UL130/UL131 pentamer,splenocytes from mice administered with CMV-gB andCMV-gH/gL/UL128/UL130/UL131 pentamer formulated with PAA adjuvant werealso ex-vivo stimulated with 15-mer peptides pools covering the sequenceof each individual protein constituting the pentamer, i.e. gH, gL,UL128, UL130 and UL131. As depicted in FIG. 13 panel B, sustainedspecific IFN-γ cellular response was detected for all the peptides poolscovering the sequence of each individual protein constituting thepentamer except UL128 for which the detected IFN-γ cellular response waslow for most of the tested mice.

In conclusion, the addition of the pentamer on the top of gB allows toincrease the IFN-γ cellular response by broadening the number ofcellular epitopes.

The invention claimed is:
 1. A subunit vaccine comprising an immunogeniccomposition consisting of: a purified protein HCMV gB antigen; apurified protein HCMV gH/gL/UL128/UL130/UL131 pentameric complexantigen; and a Th1-inducing adjuvant, wherein said Th1-inducing adjuvantcomprises a TLR4 agonist or a linear or branched polyacrylic acidpolymer salt with a weight average molecular weight Mw in the range of350 to 650 kDa.
 2. The subunit vaccine according to claim 1, whereinsaid Th1-inducing adjuvant induces in mice a lower IgG1:IgG2a,c ratio,and/or a higher INF-y level, and/or a lower IL-5 level than MF59 in acomposition comprising the same purified protein HCMV gB antigen and thesame purified protein HCMV gH/gL/UL128/UL130/UL131 pentameric complexantigen.
 3. The subunit vaccine according to claim 1, wherein saidpurified protein HCMV gB antigen comprises one or several mutations atthe endoproteolytic cleavage site.
 4. The subunit vaccine according toclaim 1, wherein said purified protein HCMV gB antigen is a full lengthgB polypeptide, a full length gB polypeptide lacking at least a portionof a transmembrane domain of the gB polypeptide, a full length gBpolypeptide lacking at least 80% of the amino acid sequence of thetransmembrane domain, a full length gB polypeptide lacking at least aportion of an intracellular domain of the gB polypeptide, a full lengthgB polypeptide lacking at least 80% of the amino acid sequence of theintracellular domain, or a full length gB polypeptide lacking at least80% of the amino acid sequences of both the transmembrane domain and theintracellular domain.
 5. The subunit vaccine according to claim 1,wherein said purified protein HCMV gB antigen is gBdTm.
 6. The subunitvaccine according to claim 1, wherein in the said purified protein HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen, the gH antigen lacksat least a portion of a transmembrane domain of the full length gH. 7.The subunit vaccine according to claim 6, wherein said gH antigencomprises an ectodomain of the full length gH encoded by UL75 gene. 8.The subunit vaccine according to claim 1, wherein the purified proteinHCMV gB and the purified protein HCMV gH/gL/UL128/UL130/UL131 pentamericcomplex are the sole HCMV protein antigens.
 9. The subunit vaccineaccording to claim 1, wherein said vaccine increases neutralizingantibody levels and/or persistence.
 10. The subunit vaccine according toclaim 6, wherein in the purified protein HCMV gH/gL/UL128/UL130/UL131pentameric complex antigen, the gH antigen lacks at least 80% of theamino acid sequence corresponding to the transmembrane domain.
 11. Asubunit vaccine comprising an immunogenic composition consisting of: apurified protein HCMV gB antigen; a purified protein HCMVgH/gL/UL128/UL130/UL131 pentameric complex antigen; and a TLR4 agonist.12. The subunit vaccine according to claim 11, wherein said TLR4 agonistis in combination with a delivery system.
 13. The subunit vaccineaccording to claim 12, wherein the delivery system is selected from thegroup consisting of aqueous nanosuspension, calcium phosphate,liposomes, virosomes, ISCOMs, micro- and nanoparticles, and emulsions.14. The subunit vaccine according to claim 11, wherein said TLR-4agonist is selected from the group consisting of: a lipopolysaccharide,a monophosphoryl lipid A (MPL), a 3-de-O-acylated monophosphoryl lipid A(3D-MPL), a glucopyranosyl lipid adjuvant (GLA), a second-generationLipid Adjuvant (SLA), a phospholipid dimer connected by anoncarbohydrate backbone and an aminoalkyl glucosaminide phosphate, or aderivative thereof.
 15. The subunit vaccine according to claim 12,wherein TLR-4 agonist in combination with a delivery system is AS01 orAS02.
 16. The subunit vaccine according to claim 11, wherein said TLR-4agonist is GLA (CAS Number 1246298-63-4) TLR-4 agonist.
 17. A subunitvaccine comprising an immunogenic composition consisting of: a purifiedprotein HCMV gB antigen; a purified protein HCMV gH/gL/UL128/UL130/UL131pentameric complex antigen; and a linear or branched polyacrylic acidpolymer salt with a weight average molecular weight Mw in the range of350 to 650 kDa.
 18. The subunit vaccine according to claim 17, whereinsaid linear or branched polyacrylic acid polymer salt is PAA225000. 19.A method of preventing a disease associated with HCMV infection in apatient in need thereof, comprising the administration of animmunologically effective amount of a subunit vaccine comprising animmunogenic composition consisting of: a purified protein HCMV gBantigen; a purified protein HCMV gH/gL/UL128/UL130/UL131 pentamericcomplex antigen; and a TLR4 agonist or a linear or branched polyacrylicacid polymer salt with a weight average molecular weight Mw in the rangeof 350 to 650 kDa, thereby preventing the disease associated with HMCVinfection in the patient.
 20. A method of producing a subunit vaccinecomprising an immunogenic composition, the method comprising: providingpurified protein antigens consisting of a purified protein HCMV gBantigen and a purified protein HCMV gH/gL/UL128/UL130/UL131 pentamericcomplex antigen; and combining the purified protein antigens with a TLR4agonist, to thereby produce an immunogenic composition.